Genetic markers associated with drought tolerance in maize

ABSTRACT

The presently disclosed subject matter relates to methods and compositions for identifying, selecting, and/or producing drought tolerant maize plants or germplasm. Maize plants or germplasm that have been identified, selected, and/or produced by any of the methods of the presently disclosed subject matter are also provided.

CROSS REFERENCES TO RELATED APPLICATIONS

The presently disclosed subject matter is a divisional of U.S. patentapplication Ser. No. 12/977,966 filed Dec. 23, 2010 and claims thebenefit of U.S. Provisional Patent Application Ser. No. 61/289,718,filed Dec. 23, 2009; and U.S. Provisional Patent Application Ser. No.61/369,999, filed Aug. 2, 2010; the disclosure of each of which isincorporated herein by reference in its entirety.

STATEMENT REGARDING ELECTRONIC SUBMISSION OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R.§1.821, entitled “72670-US-PX-D-NAT-2_Sequence_Listing_ST25”, 195kilobytes and was filed with U.S. patent application Ser. No. 12/977,966filed Dec. 23, 2009 which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/289,718, filed Dec. 23, 2009; and U.S.Provisional Patent Application Ser. No. 61/369,999, filed Aug. 2, 2010;for which this application claims the benefit thereof. The SequenceListing is attached and filed herewith and is incorporated herein byreference.

TECHNICAL FIELD

The presently disclosed subject matter relates to maize, such as maizeof the species Zea mays, and methods of breeding the same. Moreparticularly, the presently disclosed subject matter relates to maizelines, such as Zea mays lines, with one or more improved wateroptimization genotypes, and methods for breeding the same, which methodsinvolve in some embodiments genetic marker analysis and/or nucleic acidsequence analysis.

BACKGROUND

Drought is one of the major limitations to maize productionworldwide—15% of the world's maize crop is lost every year to drought.Periods of drought stress can occur at any time during the growingseason, but maize is particularly sensitive to drought stress in theperiod just before and during flowering. When drought stress occursduring this critical period, a significant decrease in grain yield canresult.

Identifying genes that enhance the drought tolerance of maize could leadto more efficient crop production by allowing for the identification,selection and production of maize plants with enhanced droughttolerance.

As such, a goal of plant breeding is to combine, in a single plant,various desirable traits. For field crops such as corn, these traits caninclude greater yield and better agronomic quality. However, geneticloci that influence yield and agronomic quality are not always known,and even if known, their contributions to such traits are frequentlyunclear. Thus, new loci that can positively influence such desirabletraits need to be identified and/or the abilities of known loci to do soneed to be discovered.

Once discovered, these desirable loci can be selected for as part of abreeding program in order to generate plants that carry desirabletraits. An exemplary embodiment of a method for generating such plantsincludes the transfer by introgression of nucleic acid sequences fromplants that have desirable genetic information into plants that do notby crossing the plants using traditional breeding techniques.

Desirable loci can be introgressed into commercially available plantvarieties using marker-assisted selection (MAS) or marker-assistedbreeding (MAB). MAS and MAB involves the use of one or more of themolecular markers for the identification and selection of those progenyplants that contain one or more loci that encode the desired traits.Such identification and selection can be based on selection ofinformative markers that are associated with desired traits. MAB canalso be used to develop near-isogenic lines (NIL) harboring loci ofinterest, allowing a more detailed study of the effect each locus canhave on a desired trait, and is also an effective method for developmentof backcross inbred line (BIL) populations.

Maize drought is one of the major limitations to maize productionworldwide. When drought stress occurs just before or during theflowering period, an increase in the length of the anthesis-silkinginterval and a decrease in grain yield can result. 15% of the world'smaize crop, or in excess of 19 millions tons, is lost every year todrought. Identifying candidate genes that can enhance drought-stresstolerance in maize could lead to more efficient crop production inaffected areas.

What are needed, then, are new methods and compositions for geneticallyanalyzing Zea mays varieties with respect to drought tolerance and foremploying the information obtained for producing new Zea mays plantsthat have improved water optimization traits.

SUMMARY

This summary lists several embodiments of the presently disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

Compositions and methods for identifying, selecting and producing maizeplants with enhanced drought tolerance are provided. A drought tolerantmaize plant or germplasm is also provided.

In some embodiments, methods of identifying a drought tolerant maizeplant or germplasm are provided. Such methods can comprise detecting, inthe maize plant or germplasm, a marker associated with enhanced droughttolerance.

In some embodiments, methods of producing a drought tolerant maize plantare provided. Such methods can comprise detecting, in a maize germplasm,the presence of a marker associated with enhanced drought tolerance andproducing a progeny plant from said maize germplasm.

In some embodiments, the presence of a marker associated with enhanceddrought tolerance is detected using a marker probe. In some suchembodiments, the presence of a marker associated with enhanced droughttolerance is detected in an amplification product from a nucleic acidsample isolated from a maize plant or germplasm. In some embodiments,the marker comprises a haplotype, and a plurality of probes is used todetect the alleles that make up the haplotype. In some such embodiments,the alleles that make up the haplotype are detected in a plurality ofamplification products from a nucleic acid sample isolated from a maizeplant or germplasm.

In some embodiments, methods of selecting a drought tolerant maize plantor germplasm are provided. Such methods can comprise crossing a firstmaize plant or germplasm with a second maize plant or germplasm, whereinthe first maize plant or germplasm comprises a marker associated withenhanced drought tolerance, and selecting a progeny plant or germplasmthat possesses the marker.

In some embodiments, methods of introgressing an allele associated withenhanced drought tolerance into a maize plant or germplasm are provided.Such methods can comprise crossing a first maize plant or germplasmcomprising an allele associated with enhanced drought tolerance with asecond maize plant or germplasm that lacks said allele and repeatedlybackcrossing progeny plants comprising said allele with the second maizeplant or germplasm to produce a drought tolerant maize plant orgermplasm comprising the allele associated with enhanced droughttolerance. Progeny comprising the allele associated with enhanceddrought tolerance can be identified by detecting, in their genomes, thepresence of a marker associated with said allele.

Maize plants and/or germplasms identified, produced or selected by anyof the methods of the invention are also provided, as are any progeny orseeds derived from a maize plant or germplasm identified, produced orselected by these methods.

Non-naturally occurring maize plants and/or germplasms comprising one ormore markers associated with enhanced drought tolerance are alsoprovided.

Isolated and/or purified markers associated with enhanced droughttolerance are also provided. Such markers can comprise a nucleotidesequence at least 85%, 90%, 95%, or 99% identical to any of SEQ ID NOs:1-117, 400, and 401, the reverse complement thereof, or an informativeor functional fragment thereof.

Compositions comprising a primer pair capable of amplifying a nucleicacid sample isolated from a maize plant or germplasm to generate amarker associated with enhanced drought tolerance are also provided.Such compositions can comprise, consist essentially of, or consist ofone of the amplification primer pairs identified in Table 1.

TABLE 1 SEQ ID NOs. of Exemplary Oligonucleotide Primers that can beEmployed for Analyzing Water Optimization Loci, Alleles, and HaplotypesGenomic Exemplary Exemplary Locus Amplification Primers Assay Primers 1, 61 118, 119 232, 233  2, 63 120, 121 346, 347; 348, 349  3, 63 122,123 234, 235  4, 64 124, 125 236, 237  5, 65 126, 127 238, 239  6, 66128, 129 240, 241  7, 67 130, 131 242, 243; 244, 245; 246, 247; 248,249; 250, 251; 350, 351; 352, 353;  8, 68 132, 133 252, 253  9, 69 134,135 254, 255 10, 70 136, 137 256, 257 11, 71 138, 139 258, 259 12, 13,72 140, 141 260, 261; 262, 263; 264, 265; 266, 267 14, 73 142, 143 268,269 15, 74 144, 145 270, 271 16, 75 146, 147 272, 273 17, 76 148, 149274, 275 18, 77 150, 151 276, 277 19, 78 152, 153 278, 279; 280, 281;282, 283; 354, 355; 356, 357 20, 79 154, 155 284, 285 21, 80 156, 157286, 287 22, 81 158, 159 288, 289 23, 82 160, 161 358, 359; 360, 361 24,83 162, 163 362, 363 25, 84 164, 165 290, 291; 364, 365 26, 85 166, 167366, 367 27, 86 168, 169 292, 293 368, 369 28, 87 170, 171 294, 295 29,88 172, 173 370, 371 30, 89 174, 175 296, 297; 298, 299 31, 90 176, 177300, 301 32, 91 178, 179 302, 303 33, 92 180, 181 372, 373 34, 93 182,183 304, 305; 306, 307; 308, 309 35, 94 184, 185 310, 311 36, 95 186,187 312, 313 37, 96 188, 189 314, 315; 316, 317 38, 97 190, 191 318,319; 320, 321 39, 98 192, 193 322, 323 40, 99 194, 195 324, 325  41, 100196, 197 326, 327; 328, 329  42, 101 198, 199 330, 331  43, 102 200, 201332, 333 44, 45, 103 202, 203 374, 375; 376, 377  46, 104 204, 205 378,379  47, 105 206, 207 380, 381  48, 106 208, 209 382, 383  49, 107 210,211 334, 335 50, 51, 108 212, 213 336, 337; 384, 385  52, 109 214, 215338, 339  53, 110 216, 217 340, 341  54, 111 218, 219 344, 345  55, 112220, 221 386, 387  56, 113 222, 223 388, 389; 390, 391  57, 114 224, 225392, 393  58, 115 226, 227 394, 395  59, 116 228, 229 396, 397  60, 117230, 231 398, 399 400, 401 402, 407 408, 409; 410, 411; 412, 413

A marker associated with enhanced drought tolerance can comprise,consist essentially of, and/or consist of a single allele or acombination of alleles at one or more genetic loci.

Thus, in some embodiments the presently disclosed subject matterprovides methods for producing a hybrid plant with enhanced wateroptimization. In some embodiments, the methods comprise (a) providing afirst plant comprising a first genotype comprising any one of haplotypesA-M: (b) providing a second plant comprising a second genotypecomprising any one of haplotypes A-M, wherein the second plant comprisesat least one of haplotypes A-M that is not present in the first plant;(c) crossing the first plant and the second maize plant to produce an F1generation; identifying one or more members of the F1 generation thatcomprises a desired genotype comprising any combination of haplotypesA-M, wherein the desired genotype differs from both the first genotypeof (a) and the second genotype of (b), whereby a hybrid plant withenhanced water optimization is produced. In some embodiments, haplotypesA-M are defined as follows:

i. Haplotype A comprises a G nucleotide at the position that correspondsto position 115 of SEQ ID NO: 1, an A nucleotide at the position thatcorresponds to position 270 of SEQ ID NO: 1, a T nucleotide at theposition that corresponds to position 301 of SEQ ID NO: 1, and an Anucleotide at the position that corresponds to position 483 of SEQ IDNO: 1 on chromosome 8 in the first plant's genome;

ii. Haplotype B comprises a deletion at positions 4497-4498 of SEQ IDNO: 7, a G nucleotide at the position that corresponds to position 4505of SEQ ID NO: 7, a T nucleotide at the position that corresponds toposition 4609 of SEQ ID NO: 7, an A nucleotide at the position thatcorresponds to position 4641 of SEQ ID NO: 7, a T nucleotide at theposition that corresponds to position 4792 of SEQ ID NO: 7, a Tnucleotide at the position that corresponds to position 4836 of SEQ IDNO: 7, a C nucleotide at the position that corresponds to position 4844of SEQ ID NO: 7, a G nucleotide at the position that corresponds toposition 4969 of SEQ ID NO: 7, and a TCC trinucleotide at the positionthat corresponds to positions 4979-4981 of SEQ ID NO: 7 on chromosome 8in the first plant's genome;

iii. Haplotype C comprises an A nucleotide at the position thatcorresponds to position 217 of SEQ ID NO: 8, a G nucleotide at theposition that corresponds to position 390 of SEQ ID NO: 8, and an Anucleotide at the position that corresponds to position 477 of SEQ IDNO: 8 on chromosome 2 in the first plant's genome;

iv. Haplotype D comprises a G nucleotide at the position thatcorresponds to position 182 of SEQ ID NO: 19, an A nucleotide at theposition that corresponds to position 309 of SEQ ID NO: 19, a Gnucleotide at the position that corresponds to position 330 of SEQ IDNO: 19, and a G nucleotide at the position that corresponds to position463 of SEQ ID NO: 19 on chromosome 8 in the first plant's genome;

v. Haplotype E comprises a C nucleotide at the position that correspondsto position 61 of SEQ ID NO: 21, a C nucleotide at the position thatcorresponds to position 200 of SEQ ID NO: 21, and a deletion of ninenucleotides at the positions that corresponds to positions 316-324 ofSEQ ID NO: 21 on chromosome 5 in the first plant's genome;

vi. Haplotype F comprises a G nucleotide at the position thatcorresponds to position 64 of SEQ ID NO: 27 and a T nucleotide at theposition that corresponds to position 254 of SEQ ID NO: 27 on chromosome8 in the first plant's genome;

vii. Haplotype G comprises an C nucleotide at the position thatcorresponds to position 98 of SEQ ID NO: 28, a T nucleotide at theposition that corresponds to position 147 of SEQ ID NO: 28, a Cnucleotide at the position that corresponds to position 224 of SEQ IDNO: 28, and a T nucleotide at the position that corresponds to position496 of SEQ ID NO: 28 on chromosome 9 in the first plant's genome;

viii. Haplotype H comprises a T nucleotide at the position thatcorresponds to position 259 of SEQ ID NO: 30, a T nucleotide at theposition that corresponds to position 306 of SEQ ID NO: 30, an Anucleotide at the position that corresponds to position 398 of SEQ IDNO: 30, and a C nucleotide at the position that corresponds to position1057 of SEQ ID NO: 30 on chromosome 4 in the first plant's genome;

ix. Haplotype I comprises a C nucleotide at the position thatcorresponds to position 500 of SEQ ID NO: 36, a G nucleotide at theposition that corresponds to position 568 of SEQ ID NO: 36, and a Tnucleotide at the position that corresponds to position 698 of SEQ IDNO: 36 on chromosome 6 in the first plant's genome;

x. Haplotype J comprises an A nucleotide at the position thatcorresponds to position 238 of SEQ ID NO: 42, a deletion of thenucleotides that correspond to positions 266-268 of SEQ ID NO: 42, and aC nucleotide at the position that corresponds to position 808 of SEQ IDNO: 42 in the first plant's genome;

xi. Haplotype K comprises a C nucleotide at the position thatcorresponds to position 166 of SEQ ID NO: 49, and A nucleotide at theposition that corresponds to position 224 of SEQ ID NO: 49, a Gnucleotide at the position that corresponds to position 650 of SEQ IDNO: 49, and a G nucleotide at the position that corresponds to position892 of SEQ ID NO: 49 on chromosome 8 in the first plant's genome;

xii. Haplotype L comprises a C nucleotide at the positions thatcorrespond to positions 83, 428, 491, and 548 of SEQ ID NO: 53 onchromosome 9 in the first plant's genome; and

xiii. Haplotype M comprises a C nucleotide at the position thatcorresponds to position 83 in SEQ ID NO: 400, an A nucleotide at theposition that corresponds to position 119 of SEQ ID NO: 400, and a Tnucleotide at the position that corresponds to position 601 of SEQ IDNO: 400.

In some embodiments, the hybrid plant with enhanced water optimizationcomprises each of haplotypes A-M that are present in the first plant aswell as at least one additional haplotype selected from haplotypes A-Mthat is present in the second plant. In some embodiments, the firstplant is a recurrent parent comprising at least one of haplotypes A-Mand the second plant is a donor that comprises at least one ofhaplotypes A-M that is not present in the first plant. In someembodiments, the first plant is homozygous for at least two, three,four, or five of haplotypes A-M. In some embodiments, the hybrid plantcomprises at least three, four, five, six, seven, eight, or nine ofhaplotypes A-M.

In some embodiments, the identifying comprises genotyping one or moremembers of an F1 generation produced by crossing the first plant and thesecond plant with respect to each of the haplotypes A-M present ineither the first plant or the second plant.

In some embodiments, the first plant and the second plant are Zea maysplants.

In some embodiments, enhanced water optimization confers increased orstabilized yield in a water stressed environment as compared to acontrol plant. In some embodiments, the hybrid with enhanced wateroptimization can be planted at a higher crop density. In someembodiments, the hybrid with enhanced water optimization confers noyield drag when under favorable moisture levels.

The presently disclosed subject matter also provides in some embodimentshybrid Zea mays plants produced by the presently disclosed methods, or acell, tissue culture, seed, or part thereof.

The presently disclosed subject matter also provides in some embodimentsinbred Zea mays plants produced by backcrossing and/or selfing and/orproducing double haploids from the hybrid Zea mays plants disclosedherein, or a cell, tissue culture, seed, or part thereof.

The presently disclosed subject matter also provides in some embodimentsinbred or hybrid Zea mays plants, the genome of which comprises at leastthree, four, five, six, seven, eight, or nine of haplotypes A-M, whereinhaplotypes A-M are associated with water optimization and are definedherein. In some embodiments, the inbred or hybrid Zea mays plantcomprises a genome comprising Haplotypes C, D, and G; Haplotypes C, D,and L; Haplotypes C, G, and H; Haplotypes C, G, and I; Haplotypes C, I,and L; Haplotypes E, G, and I; Haplotypes F, G, and H; Haplotypes A, C,F, and G; Haplotypes C, E, H, and I; Haplotypes C, G, H, and I;Haplotypes C, H, I, and K; Haplotypes C, H, I, and L; Haplotypes E, F,G, and H; Haplotypes A, C, G, H, and I; Haplotypes B, C, D, G, and L;Haplotypes C, E, G, H, and I; Haplotypes C, G, H, I, and L; HaplotypesA, C, G, H, I, and K; Haplotypes C, E, F, G, H, I, J, K, and L;Haplotypes C, D, G, and M; Haplotypes C, D, L, and M; Haplotypes C, G,H, and M; Haplotypes C, G, I, and M; Haplotypes C, I, L, and M;Haplotypes E, G, I, and M; Haplotypes F, G, H, and M; Haplotypes A, C,F, G, and M; Haplotypes C, E, H, I, and M; Haplotypes C, G, H, I, and M;Haplotypes C, H, I, K, and M; Haplotypes C, H, I, L, and M; HaplotypesE, F, G, H, and M; Haplotypes A, C, G, H, I, and M; Haplotypes B, C, D,G, L, and M; Haplotypes C, E, G, H, I, and M; Haplotypes C, G, H, I, L,and M; Haplotypes A, C, G, H, I, K, and M; and Haplotypes C, E, F, G, H,I, J, K, L, and M. In some embodiments, the inbred or hybrid Zea maysplant is a hybrid plant that is homozygous for at least one ofHaplotypes A-M.

In some embodiments, the inbred or hybrid Zea mays plant comprises agenome comprising Haplotypes A, C, E, G, H, and I, optionally furthercomprising Haplotype M; Haplotypes B, C, D, E, F, G, H, I, and L,optionally further comprising Haplotype M; Haplotypes C, D, E, F, G, H,and L, optionally further comprising Haplotype M; Haplotypes B, C, D, G,I, and L, optionally further comprising Haplotype M; Haplotypes B, C, D,E, G, H, I, and L, optionally further comprising Haplotype M; HaplotypesC, D, E, F, G, H, I, J, K, and L, optionally further comprisingHaplotype M; Haplotypes A, C, G, H, and I, optionally further comprisingHaplotype M; Haplotypes C, E, F, G, H, and I, optionally furthercomprising Haplotype M; Haplotypes C, E, F, G, H, I, and L, optionallyfurther comprising Haplotype M; Haplotypes C, D, E, F, G, and H,optionally further comprising Haplotype M; Haplotypes D, E, F, G, and H,optionally further comprising Haplotype M; Haplotypes A, C, G, H, and I,optionally further comprising Haplotype M; Haplotypes A, C, E, G, H, I,and K, optionally further comprising Haplotype M; Haplotype C, E, G, H,I, and L, optionally further comprising Haplotype M; Haplotypes C, D, E,G, H, I, and L, optionally further comprising Haplotype M; Haplotypes B,C, D, E, G, H, I, and L, optionally further comprising Haplotype M;Haplotypes A, C, G, H, and I, optionally further comprising Haplotype M;Haplotypes A, C, G, H, I, and K, optionally further comprising HaplotypeM; Haplotypes C, G H, I, and L, optionally further comprising HaplotypeM; Haplotypes C, D, G, H, I, and L, optionally further comprisingHaplotype M; Haplotypes B, C, D, G, H, I, and L, optionally furthercomprising Haplotype M; Haplotypes A, C, E, F, G, H, and I, optionallyfurther comprising Haplotype M; Haplotypes A, C, E, F, G, H, I, and K,optionally further comprising Haplotype M; Haplotypes C, E, F, G, H, I,and L, optionally further comprising Haplotype M; Haplotypes C, D, E, F,G, H, I, and L, optionally further comprising Haplotype M; Haplotypes A,C, E, F, G, H, I, J, K, and L, optionally further comprising HaplotypeM; Haplotypes A, C, E, F, G, H, I, J, K, and L, optionally furthercomprising Haplotype M; Haplotypes C, E, F, G, H, I, J, K, and L,optionally further comprising Haplotype M; Haplotypes C, D, E, F, G, H,I, J, K, and L, optionally further comprising Haplotype M; Haplotypes B,C, D, E, F, G, H, I, J, K, and L, optionally further comprisingHaplotype M; Haplotypes A, C, E, F, G, H, and I, optionally furthercomprising Haplotype M; Haplotypes A, C, E, F, G, H, I, and K,optionally further comprising Haplotype M; Haplotypes C, E, F, G, H, I,and L, optionally further comprising Haplotype M; Haplotypes B, C, D, E,F, G, H, and L, optionally further comprising Haplotype M; Haplotypes C,E, F, G, H, I, J, K, and L, optionally further comprising Haplotype M;Haplotypes C, D, G, H, and L, optionally further comprising Haplotype M;Haplotypes C, E, F, G, H, I, and L, optionally further comprisingHaplotype M; and/or Haplotypes B, C, D, E, G, I, and L, optionallyfurther comprising Haplotype M.

The presently disclosed subject matter also provides in some embodimentshybrid or inbred Zea mays plants that have been modified to include atransgene. In some embodiments, the transgene encodes a gene productthat provides resistance to a herbicide selected from among glyphosate,Sulfonylurea, imidazolinione, dicamba, glufisinate, phenoxy proprionicacid, cycloshexome, traizine, benzonitrile, and broxynil.

The presently disclosed subject matter also provides in some embodimentsmethods for identifying Zea mays plants comprising at least one alleleassociated with water optimization as disclosed herein. In someembodiments, the methods comprise (a) genotyping at least one Zea maysplant with at least one nucleic acid marker selected from among SEQ IDNOs: 1-60 and 400; and (b) selecting at least one Zea mays plantcomprising an allele of at least one of the at least one nucleic acidmarkers that is associated with water optimization.

The presently disclosed subject matter also provides in some embodimentsZea mays plants produced by introgressing an allele of interest of alocus associated with a water optimization trait into Zea maysgermplasm. In some embodiments, the introgressing comprises (a)selecting a Zea mays plant that comprises an allele of interest of alocus associated with a water optimization trait, wherein the locusassociated with a water optimization trait comprises a nucleotidesequence that is at least 90% identical to any of SEQ ID NOs: 1-117,400, and 401; and (b) introgressing the allele of interest into Zea maysgermplasm that lacks the allele.

The presently disclosed subject matter also provides in some embodimentsmethods for identifying and/or selecting drought tolerant maize plantsor germplasm. In some embodiments, the presently disclosed methodscomprise detecting, in a maize plant or germplasm, the presence of amarker associated with enhanced drought tolerance, wherein the marker isselected from the group consisting of:

a G nucleotide at the position that corresponds to position 100 of SEQID NO: 2, a TCC trinucleotide at the position that corresponds topositions 4979-4981 of SEQ ID NO: 7, a G nucleotide at the position thatcorresponds to position 116 of SEQ ID NO: 23, an A nucleotide at theposition that corresponds to position 391 of SEQ ID NO: 33, an Anucleotide at the position that corresponds to position 472 of SEQ IDNO: 48, an A nucleotide at the position that corresponds to position 237of SEQ ID NO: 56, a T nucleotide at the position that corresponds toposition 173 of SEQ ID NO: 57, and a G nucleotide at the position thatcorresponds to position 267 of SEQ ID NO: 60;

a TCC trinucleotide at the position that corresponds to positions4979-4981 of SEQ ID NO: 7, an A nucleotide at the position thatcorresponds to position 309 of SEQ ID NO: 19, a G nucleotide at theposition that corresponds to position 562 of SEQ ID NO: 25, a Cnucleotide at the position that corresponds to position 1271 of SEQ IDNO: 26, an A nucleotide at the position that corresponds to position 266of SEQ ID NO: 44, a C nucleotide at the position that corresponds toposition 386 of SEQ ID NO: 46, an A nucleotide at the position thatcorresponds to position 472 of SEQ ID NO: 48, and a G nucleotide at theposition that corresponds to position 111 of SEQ ID NO: 51;

a G nucleotide at the position that corresponds to position 100, an Anucleotide at the position that corresponds to position 4641 of SEQ IDNO: 7, an A nucleotide at the position that corresponds to position 217of SEQ ID NO: 23, a C nucleotide at the position that corresponds toposition 746 of SEQ ID NO: 24, a C nucleotide at the position thatcorresponds to position 258 of SEQ ID NO: 29, an A nucleotide at theposition that corresponds to position 266 of SEQ ID NO: 44, a Gnucleotide at the position that corresponds to position 472 of SEQ IDNO: 48, a G nucleotide at the position that corresponds to position 193of SEQ ID NO: 55, and a C nucleotide at the position that corresponds toposition 486 of SEQ ID NO: 58;

a deletion at nucleotide at the position that corresponds to positions264-271 of SEQ ID NO: 2, an A nucleotide at the position thatcorresponds to position 4641 of SEQ ID NO: 7, an A nucleotide at theposition that corresponds to position 309 of SEQ ID NO: 19, an Anucleotide at the position that corresponds to position 391 of SEQ IDNO: 33, a G nucleotide at the position that corresponds to position 237of SEQ ID NO: 56, and a C nucleotide at the position that corresponds toposition 486 of SEQ ID NO: 58;

a TCC trinucleotide at the position that corresponds to positions4979-4981 of SEQ ID NO: 7, a G nucleotide at the position thatcorresponds to position 463 of SEQ ID NO: 19, a C nucleotide at theposition that corresponds to position 254 of SEQ ID NO: 27, an Anucleotide at the position that corresponds to position 391 of SEQ IDNO: 33, a T nucleotide at the position that corresponds to position 475of SEQ ID NO: 45, a G nucleotide at the position that corresponds toposition 193 of SEQ ID NO: 55, a C nucleotide at the position thatcorresponds to position 516 of SEQ ID NO: 56, a G nucleotide at theposition that corresponds to position 729 of SEQ ID NO: 59, and a Gnucleotide at the position that corresponds to position 267 of SEQ IDNO: 60;

an A nucleotide at the position that corresponds to position 4641 of SEQID NO: 7, a G nucleotide at the position that corresponds to position463 of SEQ ID NO: 19, a C nucleotide at the position that corresponds toposition 258 of SEQ ID NO: 29, a G nucleotide at the position thatcorresponds to position 193 of SEQ ID NO: 55, and a G nucleotide at theposition that corresponds to position 237 of SEQ ID NO: 56;

a TCC trinucleotide at the position that corresponds to positions4979-4981 of SEQ ID NO: 7, an A nucleotide at the position thatcorresponds to position 472 of SEQ ID NO: 48, an A nucleotide at theposition that corresponds to position 237 of SEQ ID NO: 56, and a Tnucleotide at the position that corresponds to position 173 of SEQ IDNO: 57;

a TCC trinucleotide at the position that corresponds to positions4979-4981 of SEQ ID NO: 7, a C nucleotide at the position thatcorresponds to position 1271 of SEQ ID NO: 26, an A nucleotide at theposition that corresponds to position 266 of SEQ ID NO: 44, a Cnucleotide at the position that corresponds to position 386 of SEQ IDNO: 46, an A nucleotide at the position that corresponds to position 472of SEQ ID NO: 48, and a G nucleotide at the position that corresponds toposition 111 of SEQ ID NO: 51;

an A nucleotide at the position that corresponds to position 4641 of SEQID NO: 7, a C nucleotide at the position that corresponds to position258 of SEQ ID NO: 29, a G nucleotide at the position that corresponds toposition 87 of SEQ ID NO: 47, a G nucleotide at the position thatcorresponds to position 472 of SEQ ID NO: 48, and a G nucleotide at theposition that corresponds to position 193 of SEQ ID NO: 55;

an A nucleotide at the position that corresponds to position 4641 of SEQID NO: 7, an A nucleotide at the position that corresponds to position309 of SEQ ID NO: 19, and a G nucleotide at the position thatcorresponds to position 237 of SEQ ID NO: 56;

a TCC trinucleotide at the position that corresponds to positions4979-4981 of SEQ ID NO: 7, a G nucleotide at the position thatcorresponds to position 463 of SEQ ID NO: 19, a T nucleotide at theposition that corresponds to position 475 of SEQ ID NO: 45, and a Gnucleotide at the position that corresponds to position 193 of SEQ IDNO: 55;

a TCC trinucleotide at the position that corresponds to positions4979-4981 of SEQ ID NO: 7;

an A nucleotide at the position that corresponds to position 4641 of SEQID NO: 7;

a TCC trinucleotide at the position that corresponds to positions4979-4981 of SEQ ID NO: 7 and a C nucleotide at the position thatcorresponds to position 386 of SEQ ID NO: 46;

an A nucleotide at the position that corresponds to position 4641 of SEQID NO: 7 and a G nucleotide at the position that corresponds to position472 of SEQ ID NO: 48,

and combinations thereof, thereby identifying and/or selecting a droughttolerant maize plant or germplasm.

Thus, it is an object of the presently disclosed subject matter toprovide methods for conveying one or more water optimization traits intomaize germplasm.

An object of the presently disclosed subject matter having been statedhereinabove, and which is achieved in whole or in part by the presentlydisclosed subject matter, other objects will become evident as thedescription proceeds as best described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical depiction of the alleles present at several lociin certain of the maize varieties used in the breeding protocolsdescribed herein.

FIG. 2 is a graphical depiction of the haplotypes of the homozygousplant lines derived from the crossing of NP2391 and CML333 (“CML333homozygous −” and “CML333 homozygous +”) and of the F1 hybrid linesderived from the crossing of each of the aforementioned homozygous lineswith NP2460 (“CML333−” and “CML333+”). Lower case letters representalleles inherited from the CML333 donor line. Upper case lettersrepresent alleles inherited from NP2391 or NP2460.

FIG. 3 is a graphical depiction of the haplotypes of the homozygousplant lines derived from the crossing of NP2391 and CML322 (“CML322homozygous −” and “CML322 homozygous +”) and of the F1 hybrid linesderived from the crossing of each of the aforementioned homozygous lineswith NP2460 (“CML322−” and “CML322+”). Lower case letters representalleles inherited from the CML322 donor line. Upper case lettersrepresent alleles inherited from NP2391 or NP2460.

FIG. 4 is a graphical depiction of the haplotypes of the homozygousplant lines derived from the crossing of NP2391 and Cateto SP VII(“Cateto homozygous −” and “Cateto homozygous +”) and of the F1 hybridlines derived from the crossing of each of the aforementioned homozygouslines with NP2460 (“Cateto−” and “Cateto+”). Lower case lettersrepresent alleles inherited from the Cateto SP VII donor line. Uppercase letters represent alleles inherited from NP2391 or NP2460.

FIG. 5 is a graphical depiction of the haplotypes of the homozygousplant lines derived from the crossing of NP2391 and Confite Morocho AYA38 (“Confite homozygous −” and “Confite homozygous +”) and of the F1hybrid lines derived from the crossing of each of the aforementionedhomozygous lines with NP2460 (“Confite−” and “Confite+”). Lower caseletters represent alleles inherited from the Confite Morocho AYA 38donor line. Upper case letters represent alleles inherited from NP2391or NP2460.

FIG. 6 is a graphical depiction of the haplotypes of the homozygousplant lines derived from the crossing of NP2391 and Tuxpeno VEN 692(“Tuxpeno homozygous −” and “Tuxpeno homozygous +”) and of the F1 hybridlines derived from the crossing of each of the aforementioned homozygouslines with NP2460 (“Tuxpeno−” and “Tuxpeno+”). Lower case lettersrepresent alleles inherited from the Tuxpeno VEN 692 donor line. Uppercase letters represent alleles inherited from NP2391 or NP2460.

For each of FIGS. 1-6, the ALLELES are as follows:

ALLELE Nucleotide Position and SEQ ID NO: 1 position 87 of SEQ ID NO: 472 position 386 of SEQ ID NO: 46 3 positions 4979-4981 of SEQ ID NO: 7 4position 4641 of SEQ ID NO: 7 5 position 472 of SEQ ID NO: 48 6 position237 of SEQ ID NO: 56 7 position 516 of SEQ ID NO: 56 8 position 266 ofSEQ ID NO: 44 9 position 475 of SEQ ID NO: 45 10 position 173 of SEQ IDNO: 57 11 position 746 of SEQ ID NO: 24 12 position 391 of SEQ ID NO: 3313 position 258 of SEQ ID NO: 29 14 position 217 of SEQ ID NO: 23 15position 116 of SEQ ID NO: 23 16 position 463 of SEQ ID NO: 19 17position 309 of SEQ ID NO: 19 18 positions 264-271 of SEQ ID NO: 2 19position 100 of SEQ ID NO: 2 20 position 486 of SEQ ID NO: 58 21position 111 of SEQ ID NO: 51 22 position 254 of SEQ ID NO: 27 23position 729 of SEQ ID NO: 59 24 position 267 of SEQ ID NO: 60 25position 562 of SEQ ID NO: 25 26 position 1271 of SEQ ID NO: 26 27position 193 of SEQ ID NO: 55

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The instant disclosure includes a plurality of nucleotide and/or aminoacid sequences. Throughout the disclosure and the accompanying sequencelisting, the WIPO Standard ST.25 (1998; hereinafter the “ST.25Standard”) is employed to identify nucleotides. This nucleotideidentification standard is summarized below:

TABLE 2 Nucleotide Naming Conventions in WIPO Standard ST.25 SymbolMeaning Symbol Meaning a a k g or t/u c c s g or c g g w a or t/u t t bg or c or t/u u u d a or g or t/u r g or a h a or c or t/u v t/u or c va or g or c m a or c n a or g or c or t/u, unknown, other, or absent

In certain instances, the accompanying Sequence Listing includes one ormore specifically identified definitions for certain nucleotidepositions as set forth in lines <220> through <223> of the correspondingSequence Listing entries. For example, whereas under the ST.25 Standardthe nucleotide “n” generally substitutes for any of a, c, g, or t, inSEQ ID NO: 2 it is noted that the sequence “nnnnnnnn” at nucleotidepositions 264-271 is defined to represent either the presence or theabsence of the nucleotide sequence “CACCAAGG”. Similarly, in SEQ ID NO:5 it is noted that the sequence “nnnn” at nucleotide positions 818-821is defined to represent either the presence or the absence of thenucleotide sequence “CGCG”. As such, whereas the ST.25 Standard is to befollowed throughout the instant specification, Statement s, and SequenceListing, certain sequences disclosed herein represent specificdepartures from the ST.25 Standard, and are noted accordingly.

Additionally, whether specifically noted or not, for each recitation of“n” in the Sequence Listing, it is understood that any individual “n”(including some or all n's in a sequence of consecutive n's) canrepresent a, c, g, t/u, unknown, or other, or can be absent. Thus,unless specifically defined to the contrary in the Sequence Listing, an“n” can in some embodiments represent no nucleotide. For example, SEQ IDNO: 7 includes a string of 52 n's between nucleotides 4549 and 4600,inclusive. It is understood that one or more of these n's can be absent,including but not limited to all 52 or any subset thereof.

SEQ ID NO: 1 is a nucleotide sequence that is associated with the wateroptimization locus ZmIga4, subsequences of which can be amplified fromchromosome 8 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 2 is a nucleotide sequence that is associated with the a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 3 is a nucleotide sequence that is associated with the wateroptimization locus ZmDr1, subsequences of which can be amplified fromthe Zea mays genome using the polymerase chain reaction withamplification primers as set forth in Table 4 below.

SEQ ID NO: 4 is a nucleotide sequence that is associated with the wateroptimization locus ZmDrA encoding a voltage-dependent anion channel,subsequences of which can be amplified from chromosome 7 of the Zea maysgenome using the polymerase chain reaction with amplification primers asset forth in Table 4 below.

SEQ ID NO: 5 is a nucleotide sequence that is associated with the wateroptimization locus ZmDr2, subsequences of which can be amplified fromchromosome 2 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 6 is a nucleotide sequence that is associated with the wateroptimization locus ZmDr3, subsequences of which can be amplified fromchromosome 2 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 7 is a nucleotide sequence that is associated with the wateroptimization locus ZmDr4, subsequences of which can be amplified fromchromosome 8 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 8 is a nucleotide sequence that is associated with a wateroptimization locus ZmMa3, subsequences of which can be amplified fromchromosome 2 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 9 is a nucleotide sequence that is associated with the wateroptimization locus ZmDr6, subsequences of which can be amplified fromchromosome 4 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 10 is a nucleotide sequence that is associated with the wateroptimization locus ZmBglcn, subsequences of which can be amplified fromchromosome 3 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 11 is a nucleotide sequence that is associated with the wateroptimization locus ZmLOC100276591, subsequences of which can beamplified from the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NOs: 12 and 13 are nucleotide sequences that are associated withthe water optimization locus ZmDr7, subsequences of which can beamplified from chromosome 1 of the Zea mays genome using the polymerasechain reaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 14 is a nucleotide sequence that is associated with the wateroptimization locus ZmDr8, subsequences of which can be amplified fromthe Zea mays genome using the polymerase chain reaction withamplification primers as set forth in Table 4 below.

SEQ ID NO: 15 is a nucleotide sequence that is associated with the wateroptimization locus ZmHsp70, subsequences of which can be amplified fromchromosome 1 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 16 is a nucleotide sequence that is associated with the wateroptimization locus ZmDr9, subsequences of which can be amplified fromchromosome 4 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 17 is a nucleotide sequence that is associated with the wateroptimization locus ZmDrB, subsequences of which can be amplified fromthe Zea mays genome using the polymerase chain reaction withamplification primers as set forth in Table 4 below.

SEQ ID NO: 18 is a nucleotide sequence that is associated with the wateroptimization locus ZmAdh1-1s, subsequences of which can be amplifiedfrom chromosome 1 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 19 is a nucleotide sequence that is associated with the wateroptimization locus ZmDr10, subsequences of which can be amplified fromchromosome 8 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 20 is a nucleotide sequence that is associated with the wateroptimization locus ZmDrC, subsequences of which can be amplified fromthe Zea mays genome using the polymerase chain reaction withamplification primers as set forth in Table 4 below.

SEQ ID NO: 21 is a nucleotide sequence that is associated with the wateroptimization locus ZmDr5, subsequences of which can be amplified fromchromosome 5 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 22 is a nucleotide sequence that is associated with the wateroptimization locus ZmDrD encoding a subtilisin-chymotrypsin inhibitor 2,subsequences of which can be amplified from chromosome 5 of the Zea maysgenome using the polymerase chain reaction with amplification primers asset forth in Table 4 below.

SEQ ID NO: 23 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 24 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 25 is a nucleotide sequence that is associated with the wateroptimization locus ZmDr12, subsequences of which can be amplified fromchromosome 8 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 26 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 27 is a nucleotide sequence that is associated with the wateroptimization locus ZmDrE encoding a legumin-like protein (cl2-1),subsequences of which can be amplified from chromosome 8 of the Zea maysgenome using the polymerase chain reaction with amplification primers asset forth in Table 4 below.

SEQ ID NO: 28 is a nucleotide sequence that is associated with the wateroptimization locus ZmDrF encoding a putative cellulose synthase,subsequences of which can be amplified from chromosome 9 of the Zea maysgenome using the polymerase chain reaction with amplification primers asset forth in Table 4 below.

SEQ ID NO: 29 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 30 is a nucleotide sequence that is associated with the wateroptimization locus ZmDhn2, subsequences of which can be amplified fromchromosome 4 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 31 is a nucleotide sequence that is associated with the wateroptimization locus ZmDr16, subsequences of which can be amplified fromchromosome 8 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 32 is a nucleotide sequence that is associated with the wateroptimization locus ZmDr17, subsequences of which can be amplified fromthe Zea mays genome using the polymerase chain reaction withamplification primers as set forth in Table 4 below.

SEQ ID NO: 33 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 34 is a nucleotide sequences that is associated with thewater optimization locus ZmZCN6, subsequences of which can be amplifiedfrom chromosome 4 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 35 is a nucleotide sequence that is associated with the wateroptimization locus ZmDrG, subsequences of which can be amplified fromchromosome 5 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 36 is a nucleotide sequence that is associated with the wateroptimization locus ZmDhn1, subsequences of which can be amplified fromchromosome 6 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 37 is a nucleotide sequence that is associated with the wateroptimization locus ZmDrH, subsequences of which can be amplified fromchromosome 5 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 38 is a nucleotide sequence that is associated with the wateroptimization locus ZmDrI, subsequences of which can be amplified fromchromosome 3 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 39 is a nucleotide sequence that is associated with the wateroptimization locus ZmDrJ encoding a mcm5 DNA replication factor,subsequences of which can be amplified from chromosome 5 of the Zea maysgenome using the polymerase chain reaction with amplification primers asset forth in Table 4 below.

SEQ ID NO: 40 is a nucleotide sequence that is associated with the wateroptimization locus ZmH2B1, subsequences of which can be amplified fromchromosome 4 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 41 is a nucleotide sequence that is associated with the wateroptimization locus ZmDr3, subsequences of which can be amplified fromchromosome 2 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 42 is a nucleotide sequence that is associated with a thewater optimization locus ZmDrK encoding an inorganic phosphatase,subsequences of which can be amplified from the Zea mays genome usingthe polymerase chain reaction with amplification primers as set forth inTable 4 below.

SEQ ID NO: 43 is a nucleotide sequence that is associated with wateroptimization locus ZmCat1, subsequences of which can be amplified fromchromosome 5 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NOs: 44 and 45 are nucleotide sequences that are associated witha Zea mays water optimization locus, subsequences of which can beamplified from chromosome 8 of the Zea mays genome using the polymerasechain reaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 46 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 47 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 48 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 49 is a nucleotide sequence that is associated with the wateroptimization locus ZmRIC1, subsequences of which can be amplified fromchromosome 8 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NOs: 50 and 51 are nucleotide sequences that are associated withthe water optimization locus ZmPK4, subsequences of which can beamplified from chromosome 8 of the Zea mays genome using the polymerasechain reaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 52 is a nucleotide sequence that is associated with the wateroptimization locus Zpu1, subsequences of which can be amplified fromchromosome 2 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 53 is a nucleotide sequence that is associated with the wateroptimization locus ZmDrL, subsequences of which can be amplified fromchromosome 9 of the Zea mays genome using the polymerase chain reactionwith amplification primers as set forth in Table 4 below.

SEQ ID NO: 54 is a nucleotide sequence that is associated with the wateroptimization locus ZmDrM encoding a hexose transporter, subsequences ofwhich can be amplified from chromosome 7 of the Zea mays genome usingthe polymerase chain reaction with amplification primers as set forth inTable 4 below.

SEQ ID NO: 55 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NOs: 56 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 57 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 58 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 59 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 60 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 8 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NO: 400 is a nucleotide sequence that is associated with a Zeamays water optimization locus, subsequences of which can be amplifiedfrom chromosome 4 of the Zea mays genome using the polymerase chainreaction with amplification primers as set forth in Table 4 below.

SEQ ID NOs: 61-117 and 401 are nucleotide sequences present in theGENBANK® database (available through the World Wide Web at the websitefor the National Center for Biotechnology Information (NCBI) of theUnited States National Institutes of Health) that correspond to (i.e.,come from the same chromosomal loci in Zea mays as) SEQ ID NOs: 1-60 and400. The relationships among SEQ ID NOs: 1-60 and 400 and 61-117 and 401are set forth in Table 3.

TABLE 3 GENBANK ® Database Sequences that Correspond to SEQ ID NOs: 1-60and 400 SEQ ID NO. of GENBANK ® Corresponding Corresponding SEQ ID NO.Accession No. Nucleotides* Nucleotides 1 AC214546.3 79631-80177 61 2AC206432.3 76561-76072 62 3 AC218964.2 18179-18598 63 4 AC198035.3158268-157254 64 5 AC204020.3 180680-179781 65 6 AC206638.3120959-121302 66 7 AC206220.1 197895-190521 67 8 AC213636.3 7053-6486 689 AC184130.4 28529-28053 69 10 AC186650.4 44576-75791 70 11 AC214515.346309-46830 71 12, 13 AC211214.4 215368-214930 72 14 AC199476.4103707-103339 73 15 AC213668.4 30778-29943 74 16 AC196196.4 76499-7548175 17 AC214144.3 162815-162317 76 18 AC190915.3 6402-5517 77 19AC209819.3 153562-152716 78 20 AC187243.3 135331-136145 79 21 AC203390.386249-86674 80 22 AC195458.3 170810-171228 81 23 AC201782.4 26367-2723482 24 AC218166.3 71588-72496 83 25 AC194405.3 40048-39222 84 26AC213631.3 77810-79676 85 27 AC217937.3 111822-111263 86 28 AC211740.424016-14511 87 29 AC199040.3 88703-89626 88 30 AC203943.3 104038-10289989 31 AC210725.4 219394-219870 90 32 AC231410.4 60838-60463 91 33AC195798.3 48792-47973 92 34 AC183820.4 23492-22810 93 35 AC214256.319884-20648 94 36 AC214345.3 27168-26399 95 37 AC198140.3 149518-14909796 38 AC204009.3 60314-59762 97 39 AC205343.3 136853-136242 98 40AC196429.3 5293-5956 99 41 AC206638.3 118845-119524 100 42 AC191554.329279-28345 101 43 AC197489.3 40538-39734 102 44, 45 AC212232.361043-62624 103 46 AC187869.3 65344-64604 104 47 AC212049.4 47472-46845105 48 AC194834.3 115968-117051 106 49 AC187038.3 139008-139936 107 50,51 AC212049.4 54492-53643 108 52 AC202148.4 92457-93062 109 53AC194911.4 42128-41419 110 54 AC195167.2 55324-56161 111 55 AC202530.420157-19337 112 56 AC218457.2 26390-27041 113 57 AC195989.4114536-115181 114 58 AC207558.3 122483-121881 115 59 AC204398.3137510-138350 116 60 AC211925.4 71848-71390 117 400 AC196429.3 5293-5956401 *Numbers in this column that are listed from lower to higherindicate that the GENBANK ® database entry corresponds to the nucleotidesequence from the same strand as in the corresponding sequence disclosedin SEQ ID NOs: 1-60 and 400. For those entries in which the numbers inthis column are listed from higher to lower, the nucleotide sequencedisclosed in the GENBANK ® database entry is the reverse complement ofthe nucleotide sequence of the corresponding sequence in SEQ ID NOs:1-60 and 400.

SEQ ID NOs: 61-117 and 401 have been added to the GENBANK® database bythe Genome Sequencing Center, Washington University School of Medicine,St. Louis, Mo., United States of America. As set forth in theannotations to these database entries, the sequences were part of aneffort by The Maize Sequencing Consortium to sequence the genome of Zeamays. Currently, the sequencing effort has not been completed, andvarious portions of the Zea mays genome remain unsequenced and/or thesequences have not been ordered (or potentially, have been misordered)in the GENBANK® database.

Table 4 lists SEQ ID NOs. for oligonucleotides that can be employed toamplify Zea mays nucleic acids derived from the loci that correspond toSEQ ID NOs: 1-117, 400, and 401 and exemplary amplicons producedthereby. Table 4 also lists the nucleotide position in each locussequence of SEQ ID NOs 1-60 of a polymorphism (in some embodiments, anSNP) that is associated with a water optimization trait, as well as thecorresponding nucleotide position for the polymorphism in each amplicon.

TABLE 4 SEQ ID NOs. for Oligonucleotides that can be Employed to Amplifyand/or Assay Zea mays Loci Corresponding to SEQ ID NOs: 1-117, 400, and401 Exemplary Exemplary Amplification SNP Position(s) Assay LocusPrimers In SEQ ID NO: Primers (SEQ ID NOs.) (SEQ ID NOs) 1-60 (SEQ IDNos)  1, 61 118 and 119 115 232, 233 270 301 483  2, 62 120 and 121 100348, 349 264-271 346, 347  3, 63 122 and 123 216 234, 235  4, 64 124 and125 503 236, 237  5, 65 126 and 127 818-821 238, 239  6, 66 128 and 129254 240, 241  7, 67 130 and 131 4497-4498 246, 247 4505 244, 245 4609352, 353 4641 248, 249 4792 250, 251 4836 242, 243 4844 350, 351 49694979-4981  8, 68 132 and 133 217 252, 253 390 477  9, 69 134 and 135 292254, 255 10, 70 136 and 137 166 256, 257 11, 71 138 and 139 148 258, 25912, 13, 72 140 and 141 94 (12) 260, 261 35 (13) 262, 263 86 (13) 264,265 89 (13) 266, 267 14, 73 142 and 143 432 268, 269 15, 74 144 and 145753 270, 271 16, 75 146 and 147 755 272, 273 17, 76 148 and 149 431 274,275 18, 77 150 and 151 518 276, 277 19, 78 152 and 153 182 280, 281 309282, 283 330 356, 357 463 278, 279 354, 355 20, 79 154 and 155 773-776284, 285 21, 80 156 and 157 61 286, 287 200 316-324 22, 81 158 and 159211 288, 289 23, 82 160 and 161 116 360, 361 217 358, 359 24, 83 162 and163 746 362, 363 25, 84 164 and 165 562 290, 291 364, 365 26, 85 166 and167 1271 366, 367 27, 86 168 and 169 64 292, 293 254 368, 369 28, 87 170and 171 98 294, 295 147 224 496 29, 88 172 and 173 258 370, 371 30, 89174 and 175 259 298, 299 296 296, 297 398 1057 31, 90 176 and 177 239300, 301 32, 91 178 and 179 208 302, 303 33, 92 180 and 181 391 372, 37334, 93 182 and 183 144-145 304, 305 169 308, 309 537 306, 307 35, 94 184and 185 76 310, 311 36, 95 186 and 187 500 312, 313 568 698 37, 96 188and 189 375 316, 317 386 314, 315 38, 97 190 and 191 309 318, 319 342320, 321 39, 98 192 and 193 445 322, 323 40, 99 194 and 195 602 324, 325 41, 100 196 and 197 190 326, 327 580 328, 329  42, 101 198 and 199 238330, 331 266-268 808  43, 102 200 and 201 708 332, 333 44, 45, 103 202and 203 266 (44)  374, 375 475 (45)  376, 377  46, 104 204 and 205 386378, 379  47, 105 206 and 207 87 380, 381  48, 106 208 and 209 472 382,383  49, 107 210 and 211 166 334, 335 24 650 892 50, 51, 108 212 and 213111 (51)  384, 385 541 (50)  336, 337  52, 109 214 and 215 442 338, 339 53, 110 216 and 217 83 342, 343 428 340, 341 491 548  54, 111 218 and219 126 344, 345  55, 112 220 and 221 193 386, 387  56, 113 222 and 223237 388, 389 516 390, 391  57, 114 224 and 225 173 392, 393  58, 115 226and 227 486 394, 395  59, 116 228 and 229 729 396, 397  60, 117 230 and231 267 398, 399 400, 401 402, 403; 83 408, 409; 404, 405; 119 410, 411;406, 407 601 412, 413

As can be seen in Tables 3 and 4, certain of the sequences of SEQ IDNOs: 1-399 are related to each other. By way of example, SEQ ID NO: 1 isa nucleotide sequence from Zea mays that has been mapped to the Zea maysZmIga4 locus on chromosome 8. A subsequence of SEQ ID NO: 1 can beamplified in an amplification reaction (e.g., a PCR reaction) usingoligonucleotides having the sequences set forth in SEQ ID NOs: 118 and119 to yield an amplicon. At position 270 of SEQ ID NO: 1 there is anSNP, and the specific nucleotide that is present in any nucleic acidsample at this position can be determined using oligonucleotides thathave the sequences set forth in SEQ ID NOs: 232 and 233.

Additionally, GENBANK® Accession No. AC214546.3 includes a subsequence(i.e., nucleotides 79,631-80,177; SEQ ID NO: 61) that itself is highlysimilar to SEQ ID NO: 1 (i.e., 538/552 nucleotides identical; 98%) andthus is present at the same locus from which SEQ ID NO: 1 is derived.The differences between the two sequences (which can be identified usinga BLAST algorithm, a ClustalX algorithm, or any other appropriate methodof analysis) can be attributable to normal variation within Zea mayspopulations. A subsequence of SEQ ID NO: 61 can also be amplified in anamplification reaction (e.g., a PCR reaction) using oligonucleotideshaving the sequences set forth in SEQ ID NOs: 118 and 119 to yield anamplicon. Oligonucleotides with the sequences set forth in SEQ ID NOs:232 and 233 can also be used to assay the base that is present at theposition that corresponds to position 270 of SEQ ID NO: 1.

For SEQ ID NOs: 2-399, similar interrelationships exist with SEQ ID NOs:as are described hereinabove, and would be identifiable by one ofordinary skill in the art using routine sequence analysis techniques. Itis noted that with respect to certain of SEQ ID NOs: 1-60 and 400, thecomplete nucleotide sequence of a genomic clone that includes the fulllength sequence that corresponds to these sequences might not been yetbeen added to the GENBANK® database by The Maize Sequencing Consortium.Nonetheless, with the sequence information disclosed herein, one ofordinary skill in the art can unambigously identify the Zea mays locithat correspond to SEQ ID NOs: 1-117.

DETAILED DESCRIPTION

The presently disclosed subject matter provides compositions and methodsfor identifying, selecting, and/or producing maize plants with enhanceddrought tolerance (also referred to herein as water optimization), aswell as maize plants identified, selected and/or produced by a method ofthis invention. In addition, the presently disclosed subject matterprovides maize plants and/or germplasms having within their genomes oneor more markers associated with enhanced drought tolerance.

To assess the value of alleles and/or haplotypes under drought stress,diverse germplasm was screened in controlled field-experimentscomprising a full irrigation control treatment and a limited irrigationtreatment. A goal of the full irrigation treatment was to ensure thatwater did not limit the productivity of the crop. In contrast, a goal ofthe limited irrigation treatment was to ensure that water became themajor limiting constraint to grain yield. Main effects (e.g., treatmentand genotype) and interactions (e.g., genotype×treatment) could bedetermined when the two treatments were applied adjacent to one anotherin the field. Moreover, drought related phenotypes could be quantifiedfor each genotype in the panel thereby allowing for marker:traitassociations to be conducted.

In practice, the method for the limited irrigation treatment can varywidely depending upon the germplasm being screened, the soil type,climatic conditions at the site, pre-season water supply, and in-seasonwater supply, to name just a few. Initially, a site is identified wherein-season precipitation is low (to minimize the chance of unintendedwater application) and is suitable for cropping. In addition,determining the timing of the stress can be important, such that atarget is defined to ensure that year-to-year, or location-to-location,screening consistency is in place. An understanding of the treatmentintensity, or in some cases the yield loss desired from the limitedirrigation treatment, can also be considered. Selection of a treatmentintensity that is too light can fail to reveal genotypic variation.Selection of a treatment intensity that is too heavy can create largeexperimental error. Once the timing of stress is identified andtreatment intensity is described, irrigation can be managed in a mannerthat is consistent with these targets.

I. DEFINITIONS

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. References to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques that would be apparent to one of skill in the art.While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. For example, the phrase “a marker” refers to one or moremarkers. Similarly, the phrase “at least one”, when employed herein torefer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more of that entity,including but not limited to whole number values between 1 and 100 andgreater than 100.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about”. The term “about”, as used herein when referring to ameasurable value such as an amount of mass, weight, time, volume,concentration or percentage is meant to encompass variations of in someembodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, insome embodiments ±1%, in some embodiments ±0.5%, and in some embodiments±0.1% from the specified amount, as such variations are appropriate toperform the disclosed methods. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in this specification andattached claims are approximations that can vary depending upon thedesired properties sought to be obtained by the presently disclosedsubject matter.

As used herein, the term “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

As used herein, the term “allele” refers to a variant or an alternativesequence form at a genetic locus. In diploids, a single allele isinherited by a progeny individual separately from each parent at eachlocus. The two alleles of a given locus present in a diploid organismoccupy corresponding places on a pair of homologous chromosomes,although one of ordinary skill in the art understands that the allelesin any particular individual do not necessarily represent all of thealleles that are present in the species.

As used herein, the term “anthesis silk interval” (ASI) refers to thedifference between when a plant starts shedding pollen (anthesis) andwhen it begins producing silk (female). Data are collected on a per plotbasis. In some embodiments, this interval is expressed in days.

As used herein, the phrase “associated with” refers to a recognizableand/or assayable relationship between two entities. For example, thephrase “associated with a water optimization trait” refers to a trait,locus, gene, allele, marker, phenotype, etc., or the expression thereof,the presence or absence of which can influence an extent, degree, and/orrate at which a plant or a part of interest thereof that has the wateroptimization trait grows. As such, a marker is “associated with” a traitwhen it is linked to it and when the presence of the marker is anindicator of whether and/or to what extent the desired trait or traitform will occur in a plant/germplasm comprising the marker. Similarly, amarker is “associated with” an allele when it is linked to it and whenthe presence of the marker is an indicator of whether the allele ispresent in a plant/germplasm comprising the marker. For example, “amarker associated with enhanced drought tolerance” refers to a markerwhose presence or absence can be used to predict whether and/or to whatextent a plant will display a drought tolerant phenotype.

As used herein, the terms “backcross” and “backcrossing” refer to theprocess whereby a progeny plant is repeatedly crossed back to one of itsparents. In a backcrossing scheme, the “donor” parent refers to theparental plant with the desired gene or locus to be introgressed. The“recipient” parent (used one or more times) or “recurrent” parent (usedtwo or more times) refers to the parental plant into which the gene orlocus is being introgressed. For example, see Ragot, M. et al.Marker-assisted Backcrossing: A Practical Example, in TECHNIQUES ETUTILISATIONS DES MARQUEURS MOLECULAIRES LES COLLOQUES, Vol. 72, pp.45-56 (1995); and Openshaw et al., Marker-assisted Selection inBackcross Breeding, in PROCEEDINGS OF THE SYMPOSIUM “ANALYSIS OFMOLECULAR MARKER DATA,” pp. 41-43 (1994). The initial cross gives riseto the F1 generation. The term “BC1” refers to the second use of therecurrent parent, “BC2” refers to the third use of the recurrent parent,and so on. In some embodiments, a backcross is performed repeatedly,with a progeny individual of each successive backcross generation beingitself backcrossed to the same parental genotype.

A centimorgan (“cM”) is a unit of measure of recombination frequency.One cM is equal to a 1% chance that a marker at one genetic locus willbe separated from a marker at a second locus due to crossing over in asingle generation.

As used herein, the term “chromosome” is used in its art-recognizedmeaning of the self-replicating genetic structure in the cellularnucleus containing the cellular DNA and bearing in its nucleotidesequence the linear array of genes. The Zea mays chromosome numbersdisclosed herein refer to those as set forth in Perin et al., 2002,which relates to a reference nomenclature system adopted by L'institutNational da la Recherché Agronomique (INRA; Paris, France).

As used herein, the phrase “consensus sequence” refers to a sequence ofDNA built to identify nucleotide differences (e.g., SNP and Indelpolymorphisms) in alleles at a locus. A consensus sequence can be eitherstrand of DNA at the locus and states the nucleotide(s) at one or morepositions (e.g., at one or more SNPs and/or at one or more Indels) inthe locus. In some embodiments, a consensus sequence is used to designoligonucleotides and probes for detecting polymorphisms in the locus.

The term “comprising”, which is synonymous with “including”“containing”, or “characterized by”, is inclusive or open-ended and doesnot exclude additional, unrecited elements and/or method steps.“Comprising” is a term of art that means that the named elements and/orsteps are present, but that other elements and/or steps can be added andstill fall within the scope of the relevant subject matter.

As used herein, the phrase “consisting of” excludes any element, step,or ingredient not specifically recited. For example, when the phrase“consists of” appears in a clause of the body of a claim, rather thanimmediately following the preamble, it limits only the element set forthin that clause; other elements are not excluded from the claim as awhole.

As used herein, the phrase “consisting essentially of” limits the scopeof the related disclosure or claim to the specified materials and/orsteps, plus those that do not materially affect the basic and novelcharacteristic(s) of the disclosed and/or claimed subject matter. Forexample, the presently disclosed subject matter relates in someembodiments to introgressing favorable alleles and/or haplotypes intomaize plants. One locus that comprises certain favorable alleles and/orhaplotypes is represented by SEQ ID NO: 7, which includes nine (9)different polymorphisms as set forth herein, with nine differentfavorable alelles. For any given introgression effort with respect tothe genetic locus corresponding to SEQ ID NO: 7, the method can “consistessentially of” introgressing a particular favorable allele selectedfrom among these nine polymorphic locations, which means that therecited favorable allele is the only favorable allele introgressed intoa progeny genome. It is noted, however, that additional polymorphic lociwill also be introgressed into the genome, although the effects thereofmight be unknown or not of interest.

With respect to the terms “comprising”, “consisting essentially of”, and“consisting of”, where one of these three terms is used herein, thepresently disclosed and claimed subject matter can include the use ofeither of the other two terms. For example, the presently disclosedsubject matter relates in some embodiments to oligonucleotide primerscomprise any of SEQ ID NOs: 118-399 and 402-413. It is understood thatthe presently disclosed subject matter thus also encompassesoligonucleotide primers that in some embodiments consist essentially ofany of SEQ ID NOs: 118-399 and 402-113, as well as oligonucleotideprimers that in some embodiments consist of any of SEQ ID NOs: 118-399and 402-113. Similarly, it is also understood that in some embodimentsthe methods of the presently disclosed subject matter comprise the stepsthat are disclosed herein, in some embodiments the methods of thepresently disclosed subject matter consist essentially of the steps thatare disclosed, and in some embodiments the methods of the presentlydisclosed subject matter consist of the steps that are disclosed herein.

As used herein, the terms “cross” or “crossed” refer to the fusion ofgametes via pollination to produce progeny (e.g., cells, seeds orplants). The term encompasses both sexual crosses (the pollination ofone plant by another) and selfing (self-pollination, e.g., when thepollen and ovule are from the same plant). The term “crossing” refers tothe act of fusing gametes via pollination to produce progeny.

As used herein, the terms “cultivar” and “variety” refer to a group ofsimilar plants that by structural or genetic features and/or performancecan be distinguished from other varieties within the same species.

As used herein, the terms “desired allele” and “allele of interest” areused interchangeably to refer to an allele associated with a desiredtrait. In some embodiments, a “desired allele” and/or “allele ofinterest” can be associated with either an increase or a decrease of orin a given trait, depending on the nature of the desired phenotype. Insome embodiments, a “desired allele” and/or “allele of interest” can beassociated with a change in morphology, color, etc.

As used herein, the terms “drought tolerance” and “drought tolerant”refer to a plant's ability to endure and/or thrive under drought stressconditions. When used in reference to germplasm, the terms refer to theability of a plant that arises from that germplasm to endure and/orthrive under drought conditions. In general, a plant or germplasm islabeled as “drought tolerant” if it displays “enhanced droughttolerance.”

As used herein, the term “enhanced drought tolerance” refers to animprovement, enhancement, or increase in one or more water optimizationphenotypes as compared to one or more control plants (e.g., one or bothof the parents, or a plant lacking a marker associated with enhanceddrought tolerance). Exemplary water optimization phenotypes include, butare not limited to, grain yield at standard moisture percentage (YGSMN),grain moisture at harvest (GMSTP), grain weight per plot (GWTPN),percent yield recovery (PYREC), yield reduction (YRED), anthesis silkinterval (ASI) and percent barren (PB). Thus, a plant that demonstrateshigher YGSMN than one or both of its parents when each is grown underdrought stress conditions displays enhanced drought tolerance and can belabeled as “drought tolerant.”

As used herein, the terms “elite” and “elite line” refer to any linethat is substantially homozygous and has resulted from breeding andselection for desirable agronomic performance.

As used herein, the term “gene” refers to a hereditary unit including asequence of DNA that occupies a specific location on a chromosome andthat contains the genetic instruction for a particular characteristic ortrait in an organism.

A “genetic map” is a description of genetic linkage relationships amongloci on one or more chromosomes within a given species, generallydepicted in a diagrammatic or tabular form. For each genetic map,distances between loci are measured by the recombination frequenciesbetween them. Recombinations between loci can be detected using avariety of markers. A genetic map is a product of the mappingpopulation, types of markers used, and the polymorphic potential of eachmarker between different populations. The order and genetic distancesbetween loci can differ from one genetic map to another.

As used herein, the phrase “genetic marker” refers to a nucleic acidsequence (e.g., a polymorphic nucleic acid sequence) that has beenidentified as associated with a locus or allele of interest and that isindicative of the presence or absence of the locus or allele of interestin a cell or organism. Examples of genetic markers include, but are notlimited to genes, DNA or RNA-derived sequences, promoters, anyuntranslated regions of a gene, microRNAs, siRNAs, QTLs, transgenes,mRNAs, ds RNAs, transcriptional profiles, and methylation patterns.

As used herein, the term “genotype” refers to the genetic constitutionof an individual (or group of individuals) at one or more genetic loci,as contrasted with the observable and/or detectable and/or manifestedtrait (the phenotype). Genotype is defined by the allele(s) and/orhaplotype(s) of one or more known loci that the individual has inheritedfrom its parents. The term genotype can be used to refer to anindividual's genetic constitution at a single locus, at multiple loci,or more generally, the term genotype can be used to refer to anindividual's genetic make-up for all the genes in its genome. Genotypescan be indirectly characterized, e.g., using markers and/or directlycharacterized by nucleic acid sequencing.

As used herein, the term “germplasm” refers to genetic material of orfrom an individual (e.g., a plant), a group of individuals (e.g., aplant line, variety or family), or a clone derived from a line, variety,species, or culture. The germplasm can be part of an organism or cell,or can be separate from the organism or cell. In general, germplasmprovides genetic material with a specific molecular makeup that providesa physical foundation for some or all of the hereditary qualities of anorganism or cell culture. As used herein, germplasm includes cells, seedor tissues from which new plants can be grown, as well as plant parts,such as leafs, stems, pollen, or cells that can be cultured into a wholeplant.

A “haplotype” is the genotype of an individual at a plurality of geneticloci, i.e., a combination of alleles. Typically, the genetic loci thatdefine a haplotype are physically and genetically linked, i.e., on thesame chromosome segment. The term “haplotype” can refer to polymorphismsat a particular locus, such as a single marker locus, or polymorphismsat multiple loci along a chromosomal segment.

A “heterotic group” comprises a set of genotypes that perform well whencrossed with genotypes from a different heterotic group. Hallauer etal., Corn breeding, in CORN AND CORN IMPROVEMENT p. 463-564 (1998).Inbred lines are classified into heterotic groups, and are furthersubdivided into families within a heterotic group, based on severalcriteria such as pedigree, molecular marker-based associations, andperformance in hybrid combinations. Smith et al., Theor. Appl. Gen.80:833 (1990).

As used herein, the term “heterozygous” refers to a genetic statuswherein different alleles reside at corresponding loci on homologouschromosomes. As used herein, the term “homozygous” refers to a geneticstatus wherein identical alleles reside at corresponding loci onhomologous chromosomes. It is noted that both of these terms can referto single nucleotide positions, multiple nucleotide positions, whethercontiguous or not, or entire loci on homologous chromosomes.

As used herein, the term “hybrid” refers to a seed and/or plant producedwhen at least two genetically dissimilar parents are crossed.

As used herein, the term “hybrid” when used in the context of nucleicacids, refers to a double-stranded nucleic acid molecule, or duplex,formed by hydrogen bonding between complementary nucleotide bases. Theterms “hybridize” and “anneal” refer to the process by which singlestrands of nucleic acid sequences form double-helical segments throughhydrogen bonding between complementary bases.

As used herein, the phrase “ILLUMINA® GOLDENGATE® Assay” refers to ahigh throughput genotyping assay sold by Illumina Inc. of San Diego,Calif., United States of America that can generate SNP-specific PCRproducts. This assay is described in detail at the website of IlluminaInc. and in Fan et al., 2006.

As used herein, the phrase “immediately adjacent”, when used to describea nucleic acid molecule that hybridizes to DNA containing apolymorphism, refers to a nucleic acid that hybridizes to a DNA sequencethat directly abuts the polymorphic nucleotide base position. Forexample, a nucleic acid molecule that can be used in a single baseextension assay is “immediately adjacent” to the polymorphism.

As used herein, the term “improved”, and grammatical variants thereof,refers to a plant or a part, progeny, or tissue culture thereof, that asa consequence of having (or lacking) a particular water optimizationassociated allele (such as, but not limited to those water optimizationassociated alleles disclosed herein) is characterized by a higher orlower content of a water optimization associated trait, depending onwhether the higher or lower content is desired for a particular purpose.

As used herein, the term “inbred” refers to a substantially homozygousplant or variety. The term can refer to a plant or variety that issubstantially homozygous throughout the entire genome or that issubstantially homozygous with respect to a portion of the genome that isof particular interest.

As used herein, the term “INDEL” (also spelled “indel”) refers to aninsertion or deletion in a pair of nucleotide sequences, wherein a firstsequence can be referred to as having an insertion relative to a secondsequence or the second sequence can be referred to as having a deletionrelative to the first sequence.

As used herein, the term “informative fragment” refers to a nucleotidesequence comprising a fragment of a larger nucleotide sequence, whereinthe fragment allows for the identification of one or more alleles withinthe larger nucleotide sequence. For example, an informative fragment ofthe nucleotide sequence of SEQ ID NO: 1 comprises a fragment of thenucleotide sequence of SEQ ID NO: 1 and allows for the identification ofone or more alleles (e.g., a G nucleotide at position 115 of SEQ ID NO:1, an A nucleotide at the position that corresponds to position 270 ofSEQ ID NO: 1, a T nucleotide at the position that corresponds toposition 301 of SEQ ID NO: 1, and/or an A nucleotide at the positionthat corresponds to position 483).

As used herein, the phrase “interrogation position” refers to a physicalposition on a solid support that can be queried to obtain genotypingdata for one or more predetermined genomic polymorphisms.

As used herein, the terms “introgression,” “introgressing” and“introgressed” refer to both the natural and artificial transmission ofa desired allele or combination of desired alleles of a genetic locus orgenetic loci from one genetic background to another. For example, adesired allele at a specified locus can be transmitted to at least oneprogeny via a sexual cross between two parents of the same species,where at least one of the parents has the desired allele in its genome.Alternatively, for example, transmission of an allele can occur byrecombination between two donor genomes, e.g., in a fused protoplast,where at least one of the donor protoplasts has the desired allele inits genome. The desired allele can be a selected allele of a marker, aQTL, a transgene, or the like. Offspring comprising the desired allelecan be repeatedly backcrossed to a line having a desired geneticbackground and selected for the desired allele, with the result beingthat the desired allele becomes fixed in the desired genetic background.For example, a marker associated with enhanced drought tolerance can beintrogressed from a donor into a recurrent parent that is not droughttolerant or only partially drought tolerant. The resulting offspringcould then be repeatedly backcrossed and selected until the progenypossess the drought tolerance allele in the recurrent parent background.

As used herein, the term “isolated” refers to a nucleotide sequence(e.g., a genetic marker) that is free of sequences that normally flankone or both sides of the nucleotide sequence in a plant genome. As such,the phrase “isolated and purified genetic marker associated with a wateroptimization trait in Zea mays” can be, for example, a recombinant DNAmolecule, provided one of the nucleic acid sequences normally foundflanking that recombinant DNA molecule in a naturally-occurring genomeis removed or absent. Thus, isolated nucleic acids include, withoutlimitation, a recombinant DNA that exists as a separate molecule(including, but not limited to genomic DNA fragments produced by PCR orrestriction endonuclease treatment) with no flanking sequences present,as well as a recombinant DNA that is incorporated into a vector, anautonomously replicating plasmid, or into the genomic DNA of a plant aspart of a hybrid or fusion nucleic acid molecule.

As used herein, the term “linkage” refers to a phenomenon whereinalleles on the same chromosome tend to be transmitted together moreoften than expected by chance if their transmission were independent.Thus, two alleles on the same chromosome are said to be “linked” whenthey segregate from each other in the next generation in someembodiments less than 50% of the time, in some embodiments less than 25%of the time, in some embodiments less than 20% of the time, in someembodiments less than 15% of the time, in some embodiments less than 10%of the time, in some embodiments less than 9% of the time, in someembodiments less than 8% of the time, in some embodiments less than 7%of the time, in some embodiments less than 6% of the time, in someembodiments less than 5% of the time, in some embodiments less than 4%of the time, in some embodiments less than 3% of the time, in someembodiments less than 2% of the time, and in some embodiments less than1 of the time.

As such, “linkage” typically implies and can also refer to physicalproximity on a chromosome. Thus, two loci are linked if they are withinin some embodiments 20 centiMorgans (cM), in some embodiments 15 cM, insome embodiments 12 cM, in some embodiments 10 cM, in some embodiments 9cM, in some embodiments 8 cM, in some embodiments 7 cM, in someembodiments 6 cM, in some embodiments 5 cM, in some embodiments 4 cM, insome embodiments 3 cM, in some embodiments 2 cM, and in some embodiments1 cM of each other. Similarly, a yield locus of the presently disclosedsubject matter is linked to a marker (e.g., a genetic marker) if it isin some embodiments within 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1cM of the marker.

Thus, the term “linkage” refers to the degree with which one markerlocus is associated with another marker locus or some other locus (forexample, a drought tolerance locus). The linkage relationship between amolecular marker and a phenotype can be given as a “probability” or“adjusted probability.” Linkage can be expressed as a desired limit orrange. For example, in some embodiments, any marker is linked(genetically and physically) to any other marker when the markers areseparated by less than about 50, 40, 30, 25, 20, or 15 map units (orcM).

In some embodiments of the presently disclosed subject matter, it isadvantageous to define a bracketed range of linkage, for example, fromabout 10 cM and about 20 cM, from about 10 cM and about 30 cM, or fromabout 10 cM and about 40 cM. The more closely a marker is linked to asecond locus, the better an indicator for the second locus that markerbecomes. Thus, “closely linked loci” such as a marker locus and a secondlocus display an inter-locus recombination frequency of about 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, or 2% or less. In some embodiments, the relevantloci display a recombination frequency of about 1% or less, e.g., about0.75%, 0.5%, 0.25% or less. Two loci that are localized to the samechromosome, and at such a distance that recombination between the twoloci occurs at a frequency of less than about 10% (e.g., about 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, or 0.25%, or less) can also besaid to be “proximal to” each other. Since one cM is the distancebetween two markers that show a 1% recombination frequency, any markeris closely linked (genetically and physically) to any other marker thatis in close proximity, e.g., at or less than about 10 cM distant. Twoclosely linked markers on the same chromosome can be positioned about 9,8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5 or 0.25 cM or less from each other.

As used herein, the term “linkage disequilibrium” refers to a non-randomsegregation of genetic loci or traits (or both). In either case, linkagedisequilibrium implies that the relevant loci are within sufficientphysical proximity along a length of a chromosome so that they segregatetogether with greater than random (i.e., non-random) frequency (in thecase of co-segregating traits, the loci that underlie the traits are insufficient proximity to each other). Markers that show linkagedisequilibrium are considered linked. Linked loci co-segregate more than50% of the time, e.g., from about 51% to about 100% of the time. Inother words, two markers that co-segregate have a recombinationfrequency of less than 50% (and, by definition, are separated by lessthan 50 cM on the same chromosome). As used herein, linkage can bebetween two markers, or alternatively between a marker and a phenotype.A marker locus can be “associated with” (linked to) a trait, e.g.,drought tolerance. The degree of linkage of a molecular marker to aphenotypic trait is measured, e.g., as a statistical probability ofco-segregation of that molecular marker with the phenotype.

Linkage disequilibrium is most commonly assessed using the measure r²,which is calculated using the formula described by Hill and Robertson,Theor. Appl. Genet. 38:226 (1968). When r²=1, complete linkagedisequilibrium exists between the two marker loci, meaning that themarkers have not been separated by recombination and have the sameallele frequency. Values for r² above ⅓ indicate sufficiently stronglinkage disequilibrium to be useful for mapping. Ardlie et al., NatureReviews Genetics 3:299 (2002). Hence, alleles are in linkagedisequilibrium when r² values between pairwise marker loci are greaterthan or equal to about 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.

As used herein, the term “linkage equilibrium” describes a situationwhere two markers independently segregate, i.e., sort among progenyrandomly. Markers that show linkage equilibrium are considered unlinked(whether or not they lie on the same chromosome). As such, the phrase“linkage disequilibrium” is defined as change from the expected relativefrequency of gamete types in a population of many individuals in asingle generation such that two or more loci act as genetically linkedloci. If the frequency in a population of allele S is x, s is x′, B isy, and b is y′, then the expected frequency of genotype SB is xy, thatof Sb is xy′, that of sB is x′y, and that of sb is x′y′, and anydeviation from these frequencies is an example of disequilibrium.

As used herein, the phrase “linkage group” refers to all of the genes orgenetic traits that are located on the same chromosome. Within thelinkage group, those loci that are close enough together can exhibitlinkage in genetic crosses. Since the probability of crossover increaseswith the physical distance between loci on a chromosome, loci for whichthe locations are far removed from each other within a linkage groupmight not exhibit any detectable linkage in direct genetic tests. Theterm “linkage group” is mostly used to refer to genetic loci thatexhibit linked behavior in genetic systems where chromosomal assignmentshave not yet been made. Thus, in the present context, the term “linkagegroup” is synonymous with the physical entity of a chromosome, althoughone of ordinary skill in the art will understand that a linkage groupcan also be defined as corresponding to a region of (i.e., less than theentirety) of a given chromosome.

A “locus” is a position on a chromosome where a gene or marker or alleleis located. In some embodiments, a locus can encompass one or morenucleotides.

As used herein, the term “maize” refers to a plant of the Zea mays L.ssp. mays and is also known as “corn.”

As used herein, the term “maize plant” includes whole maize plants,maize plant cells, maize plant protoplast, maize plant cell or maizetissue cultures from which maize plants can be regenerated, maize plantcalli, and maize plant cells that are intact in maize plants or parts ofmaize plants, such as maize seeds, maize cobs, maize flowers, maizecotyledons, maize leaves, maize stems, maize buds, maize roots, maizeroot tips, and the like.

As used herein, the terms “marker”, “genetic marker”, and “molecularmarker” are used interchangeably to refer to an identifiable position ona chromosome the inheritance of which can be monitored and/or a reagentthat is used in methods for visualizing differences in nucleic acidsequences present at such identifiable positions on chromosomes. Thus,in some embodiments a marker comprises a known or detectable nucleicacid sequence. Examples of markers include, but are not limited togenetic markers, protein composition, peptide levels, protein levels,oil composition, oil levels, carbohydrate composition, carbohydratelevels, fatty acid composition, fatty acid levels, amino acidcomposition, amino acid levels, biopolymers, starch composition, starchlevels, fermentable starch, fermentation yield, fermentation efficiency(e.g., captured as digestibility at 24, 48, and/or 72 hours), energyyield, secondary compounds, metabolites, morphological characteristics,and agronomic characteristics. As such, a marker can comprise anucleotide sequence that has been associated with an allele or allelesof interest and that is indicative of the presence or absence of theallele or alleles of interest in a cell or organism and/or to a reagentthat is used to visualize differences in the nucleotide sequence at suchan identifiable position or positions. A marker can be, but is notlimited to, an allele, a gene, a haplotype, a restriction fragmentlength polymorphism (RFLP), a simple sequence repeat (SSR), randomamplified polymorphic DNA (RAPD), cleaved amplified polymorphicsequences (CAPS) (Rafalski and Tingey, Trends in Genetics 9:275 (1993)),an amplified fragment length polymorphism (AFLP) (Vos et al., NucleicAcids Res. 23:4407 (1995)), a single nucleotide polymorphism (SNP)(Brookes, Gene 234:177 (1993)), a sequence-characterized amplifiedregion (SCAR) (Paran and Michelmore, Theor. Appl. Genet. 85:985 (1993)),a sequence-tagged site (STS) (Onozaki et al., Euphytica 138:255 (2004)),a single-stranded conformation polymorphism (SSCP) (Orita et al., Proc.Natl. Acad. Sci. USA 86:2766 (1989)), an inter-simple sequence repeat(ISSR) (Blair et al., Theor. Appl. Genet. 98:780 (1999)), aninter-retrotransposon amplified polymorphism (IRAP), aretrotransposon-microsatellite amplified polymorphism (REMAP) (Kalendaret al., Theor. Appl. Genet. 98:704 (1999)) or an RNA cleavage product(such as a Lynx tag). A marker can be present in genomic or expressednucleic acids (e.g., ESTs). The term marker can also refer to nucleicacids used as probes or primers (e.g., primer pairs) for use inamplifying, hybridizing to and/or detecting nucleic acid moleculesaccording to methods well known in the art. A large number of maizemolecular markers are known in the art, and are published or availablefrom various sources, such as the Maize GDB internet resource and theArizona Genomics Institute internet resource run by the University ofArizona.

In some embodiments, a marker corresponds to an amplification productgenerated by amplifying a Zea mays nucleic acid with one or moreoligonucleotides, for example, by the polymerase chain reaction (PCR).As used herein, the phrase “corresponds to an amplification product” inthe context of a marker refers to a marker that has a nucleotidesequence that is the same (allowing for mutations introduced by theamplification reaction itself and/or naturally occurring and/orartificial alleleic differences) as an amplification product that isgenerated by amplifying Zea mays genomic DNA with a particular set ofoligonucleotides. In some embodiments, the amplifying is by PCR, and theoligonucleotides are PCR primers that are designed to hybridize toopposite strands of the Zea mays genomic DNA in order to amplify a Zeamays genomic DNA sequence present between the sequences to which the PCRprimers hybridize in the Zea mays genomic DNA. The amplified fragmentthat results from one or more rounds of amplification using such anarrangement of primers is a double stranded nucleic acid, one strand ofwhich has a nucleotide sequence that comprises, in 5′ to 3′ order, thesequence of one of the primers, the sequence of the Zea mays genomic DNAlocated between the primers, and the reverse-complement of the secondprimer. Typically, the “forward” primer is assigned to be the primerthat has the same sequence as a subsequence of the (arbitrarilyassigned) “top” strand of a double-stranded nucleic acid to beamplified, such that the “top” strand of the amplified fragment includesa nucleotide sequence that is, in 5′ to 3′ direction, equal to thesequence of the forward primer—the sequence located between the forwardand reverse primers of the top strand of the genomic fragment—thereverse-complement of the reverse primer. Accordingly, a marker that“corresponds to” an amplified fragment is a marker that has the samesequence of one of the strands of the amplified fragment.

Markers corresponding to genetic polymorphisms between members of apopulation can be detected by methods well-established in the art. Theseinclude, e.g., nucleic acid sequencing, hybridization methods,amplification methods (e.g., PCR-based sequence specific amplificationmethods), detection of restriction fragment length polymorphisms (RFLP),detection of isozyme markers, detection of polynucleotide polymorphismsby allele specific hybridization (ASH), detection of amplified variablesequences of the plant genome, detection of self-sustained sequencereplication, detection of simple sequence repeats (SSRs), detection ofsingle nucleotide polymorphisms (SNPs), and/or detection of amplifiedfragment length polymorphisms (AFLPs). Well established methods are alsoknown for the detection of expressed sequence tags (ESTs) and SSRmarkers derived from EST sequences and randomly amplified polymorphicDNA (RAPD).

A “marker allele,” also described as an “allele of a marker locus,” canrefer to one of a plurality of polymorphic nucleotide sequences found ata marker locus in a population that is polymorphic for the marker locus.

As used herein, the phrase “marker assay” refers to a method fordetecting a polymorphism at a particular locus using a particular methodsuch as but not limited to measurement of at least one phenotype (suchas seed color, oil content, or a visually detectable trait); nucleicacid-based assays including, but not limited to restriction fragmentlength polymorphism (RFLP), single base extension, electrophoresis,sequence alignment, allelic specific oligonucleotide hybridization(ASO), random amplified polymorphic DNA (RAPD), microarray-basedtechnologies, TAQMAN® Assays, ILLUMINA® GOLDENGATE® Assay analysis,nucleic acid sequencing technologies; peptide and/or polypeptideanalyses; or any other technique that can be employed to detect apolymorphism in an organism at a locus of interest.

“Marker-assisted selection” (MAS) is a process by which phenotypes areselected based on marker genotypes.

“Marker-assisted counter-selection” is a process by which markergenotypes are used to identify plants that will not be selected,allowing them to be removed from a breeding program or planting.

As used herein, the terms “marker locus” and “marker loci” refer to aspecific chromosome location or locations in the genome of an organismwhere a specific marker or markers can be found. A marker locus can beused to track the presence of a second linked locus, e.g., a linkedlocus that encodes or contributes to expression of a phenotypic trait.For example, a marker locus can be used to monitor segregation ofalleles at a locus, such as a QTL or single gene, that are geneticallyor physically linked to the marker locus.

As used herein, the terms “marker probe” and “probe” refer to anucleotide sequence or nucleic acid molecule that can be used to detectthe presence of one or more particular alleles within a marker locus(e.g., a nucleic acid probe that is complementary to all of or a portionof the marker or marker locus, through nucleic acid hybridization).Marker probes comprising about 8, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100 or more contiguous nucleotides can be used for nucleic acidhybridization. Alternatively, in some aspects, a marker probe refers toa probe of any type that is able to distinguish (i.e., genotype) theparticular allele that is present at a marker locus.

As used herein, the term “molecular marker” can be used to refer to agenetic marker, as defined above, or an encoded product thereof (e.g., aprotein) used as a point of reference when identifying a linked locus. Amolecular marker can be derived from genomic nucleotide sequences orfrom expressed nucleotide sequences (e.g., from a spliced RNA, a cDNA,etc.). The term also refers to nucleotide sequences complementary to orflanking the marker sequences, such as nucleotide sequences used asprobes and/or primers capable of amplifying the marker sequence.Nucleotide sequences are “complementary” when they specificallyhybridize in solution, e.g., according to Watson-Crick base pairingrules. Some of the markers described herein are also referred to ashybridization markers when located on an indel region. This is becausethe insertion region is, by definition, a polymorphism vis-ã-vis a plantwithout the insertion. Thus, the marker need only indicate whether theindel region is present or absent. Any suitable marker detectiontechnology can be used to identify such a hybridization marker, e.g.,SNP technology is used in the examples provided herein.

The presently disclosed subject matter provides in some embodimentsmarkers for determining the presence of genetic polymorphisms in themaize loci disclosed herein. The loci that can be analyzed using thecompositions and methods of the presently disclosed subject matterinclude, but are not limited to the loci referred to herein as“ZmAdh1-1s”, “ZmBglcn”, “ZmCat1”, “ZmDhn1”, “ZmDhn2”, “ZmDr1”, “ZmDr2”,“ZmDr3”, “ZmDr3”, “ZmDr4”, “ZmDr5”, “ZmDr6”, “ZmDr7”, “ZmDr8”, “ZmDr9”,“ZmDr10”, “ZmDr12”, “ZmDr16”, “ZmDr17”, “ZmH2B1”, “ZmHsp70”, “ZmIga4”,“ZmLOC100276591”, “ZmMa3”, “ZmPK4”, “ZmRIC1”, “ZmZCN6”, “Zpu1”, “ZmDrA”,“ZmDrB”, “ZmDrC”, “ZmDrD”, “ZmDrE”, “ZmDrF”, “ZmDrG”, “ZmDrH”, “ZmDrI”,“ZmDrj”, “ZmDrk”, “ZmDrL”, and “ZmDrM”, which terms thus refer togenomic regions and/or genetic loci that are linked to wateroptimization associated traits present on Zea mays chromosomes and asdescribed in more detail hereinbelow. Exemplary genomic nucleotidesequences that are derived from these loci are summarized herein above.

The term “ZmAdh1-1s” refers to a locus on Zea mays chromosome 1 thatencodes a alcohol dehydrogenase 1 gene (Dennis et al., 1984). Exemplarygene products derived from the ZmAdh1-1s locus can be found in GENBANK®Accession Nos. X04049 and P00333.

The term “ZmBglcn” refers to a locus on Zea mays chromosome 3 thatencodes a maize 1,3-β-glucanase polypeptide (Wu et al., 1994). Exemplarygene products derived from the ZmBglcn locus can be found in GENBANK®Accession Nos. M95407 and AAA74320.

The term “ZmCat1” refers to a locus on Zea mays chromosome 5 thatencodes a maize catalast 1 polypeptide (Guan & Scandalios, 1993).Exemplary gene products derived from the ZmCat1 locus can be found inGENBANK® Accession Nos. X60135 and CAA42720.

The term “ZmDhn1” refers to a locus on Zea mays chromosome 6 thatencodes a maize dehydrin-1 (dhn1) polypeptide (Close et al., 1989).Exemplary gene products derived from the ZmDhn1 locus can be found inGENBANK® Accession Nos. X15290 and CAA33364.

The term “ZmDhn2” refers to a locus on Zea mays chromosome 4 thatencodes a maize dehydrin-2 (dhn2) polypeptide. Exemplary gene productsderived from the ZmDhn2 locus can be found in GENBANK® Accession Nos.L35913 and AA33480.

The term “ZmDr1” refers to a Zea mays locus that in some embodimentscorresponds to GENBANK® Accession No. AY105200.

The term “ZmDr2” refers to a Zea mays locus that in some embodimentscorresponds to GENBANK® Accession No. AF043347.

The term “ZmDr3” refers to a Zea mays locus that in some embodimentscorresponds to nucleotides 120,959-121,302 of GENBANK® Accession No.AC206638.3 and in some embodiments corresponds to GENBANK® Accession No.AF043347.

The term “ZmDr4” refers to a Zea mays locus that in some embodimentscorresponds to GENBANK® Accession No. AY103545.

The term “ZmDr5” refers to a Zea mays locus that in some embodimentscorresponds to GENBANK® Accession No. AY109606.

The term “ZmDr6” refers to a Zea mays locus that encodes a maizecalmodulin-binding protein. Exemplary gene products derived from theZmDr6 locus can be found in GENBANK® Accession Nos. L01497,NM_001158968, AAA33447, and NP_001152440.

The term “ZmDr7” refers to a Zea mays locus that encodes a maize sucrosetransporter protein. Exemplary gene products derived from the ZmDr7locus can be found in GENBANK® Accession Nos. AB008464, NM_001111370,BAA83501. and NP_001104840.

The term “ZmDr8” refers to a Zea mays locus that in some embodimentscorresponds to GENBANK® Accession No. EU976286.

The term “ZmDr9” refers to a Zea mays locus that in some embodimentscorresponds to nucleotides 75,481-76,499 of GENBANK® Accession No.AC196196.4.

The term “ZmDr10” refers to a Zea mays locus that in some embodimentscorresponds to GENBANK® Accession No. DQ245017.

The term “ZmDr12” refers to a Zea mays locus that in some embodimentscorresponds to GENBANK® Accession No. AI770817.

The term “ZmDr16” refers to a Zea mays locus that in some embodimentscorresponds to GENBANK® Accession No. NM_001156978.

The term “ZmDr17” refers to a Zea mays locus that in some embodimentscorresponds to nucleotides 60,463-60,838 of GENBANK® Accession No.AC231410.4.

The term “ZmDrA” refers to a locus on Zea mays chromosome 7 that encodesa voltage-dependent anion channel protein. An exemplary gene productderived from the ZmDrA locus can be found in GENBANK® Accession No.BT018647.

The term “ZmDrB” refers to a Zea mays locus that encodes a xylanendohydrolase protein. An exemplary gene product derived from the ZmDrBlocus can be found in GENBANK® Accession No. AI691894.

The term “ZmDrC” refers to a Zea mays locus that encodes atrehalose-P-synthase protein. An exemplary gene product derived from theZmDrC locus can be found in GENBANK® Accession No. AY110270.

The term “ZmDrD” refers to a locus on Zea mays chromosome 5 that encodesa subtilisin-chymotrypsin inhibitor 2 protein. An exemplary gene productderived from the ZmDrD locus can be found in GENBANK® Accession No.BT066886.

The term “ZmDrE” refers to a locus on Zea mays chromosome 8 that encodesa legumin-like protein (cl2-1) protein. Exemplary gene products derivedfrom the ZmDrE locus can be found in GENBANK® Accession Nos.NM_001111592 and NP_001105062.

The term “ZmDrF” refers to a locus on Zea mays chromosome 9 that encodesa putative cellulose synthase protein. Exemplary gene products derivedfrom the ZmDrF locus can be found in GENBANK® Accession Nos. BT067558and ACN34455.

The term “ZmDrG” refers to a locus on Zea mays chromosome 5 that in someembodiments corresponds to GENBANK® Accession No. AI691276.

The term “ZmDrH” refers to a locus on Zea mays chromosome 5 that in someembodiments corresponds to GENBANK® Accession No. AI665888.

The term “ZmDrI” refers to a locus on Zea mays chromosome 3 that in someembodiments corresponds to GENBANK® Accession No. AI737958.

The term “ZmDrJ” refers to a locus on Zea mays chromosome 5 that encodesa mcm5 DNA replication factor protein. An exemplary gene productsderived from the ZmDrJ locus can be found in GENBANK® Accession No.AI666237.

The term “ZmDrK” refers to a Zea mays locus that encodes an inorganicphosphatase protein that in some embodiments corresponds to nucleotides28,345-29,279 of GENBANK® Accession No. AC191554.3.

The term “ZmDrL” refers to a locus on Zea mays chromosome 9 that encodesa late embryonic abundant-like protein. An exemplary gene productderived from the ZmDrL locus can be found in GENBANK® Accession No.AY105938.

The term “ZmDrM” refers to a locus on Zea mays chromosome 7 that encodesa hexose transporter protein. Exemplary gene products derived from theZmDrM locus can be found in GENBANK® Accession Nos. NM_001154535 andNP_001148007.

The term “ZmH2B1” refers to a locus on Zea mays chromosome 4 thatencodes a Zea mays histone 2B1. An exemplary gene product derived fromthe ZmDr6 locus can be found in GENBANK® Accession No. AI737900.

The term “ZmHsp70” refers to a locus on Zea mays chromosome 1 thatencodes a maize heat shock cognate 70 kDa protein 2 protein. Exemplarygene products derived from the ZmDr7 locus can be found in GENBANK®Accession Nos. EU971059, NM_001154726, and NP_001148198.

The term “ZmIga4” refers to a locus on Zea mays chromosome 8 thatencodes a liguleless4 (lg4) protein. Exemplary gene products derivedfrom the ZmIga4 locus can be found in GENBANK® Accession Nos. AF457121,NM_001111614, AAM27190, and NP_001105084.

The term “ZmLOC100276591” refers to a locus that in some embodimentscorresponds to GENBANK® Accession Nos. NM_001150343 and NP_001143815.

The term “ZmMa3” refers to a locus on Zea mays chromosome 2 that encodesa maize topoisomerase-like apoptosis protein ma-3. Exemplary geneproducts derived from the ZmMa3 locus can be found in GENBANK® AccessionNos. NM_001154442 and NP_001147914.

The term “ZmPK4” refers to a locus on Zea mays chromosome 8 that encodesa maize protein kinase PK4 protein. Exemplary gene products derived fromthe ZmPK4 locus can be found in GENBANK® Accession Nos. AF141378,NM_001111470, AAF22219, and NP_001104940.

The term “ZmRIC1” refers to a locus on Zea mays chromosome 8 thatencodes a maize ras-related protein RIC1. Exemplary gene productsderived from the ZmRIC1 locus can be found in GENBANK® Accession Nos.EU952511, NM_001137272, ACG24629, and NP_001130744.

The term “ZmZCN6” refers to a locus on Zea mays chromosome 4 thatencodes a maize ZCN6 protein. Exemplary gene products derived from theZmZCN6 locus can be found in GENBANK® Accession Nos. EU241897,NM_001112774, ABX11008, and NP_001106245.

The term “Zpu1” refers to a locus on Zea mays chromosome 2 that encodesa pullulanase-type starch debranching enzyme (zpu1) protein. Exemplarygene products derived from the Zpu1 locus can be found in GENBANK®Accession Nos. AF080567, NM_001111450, AAD11599, and NP_001104920.

As used herein, the phrase “native trait” refers to any existingmonogenic or oligogenic trait in a certain crop's germplasm. Whenidentified through molecular marker(s), the information obtained can beused for the improvement of germplasm through marker assisted breedingof the water optimization associated traits disclosed herein.

A “non-naturally occurring variety of maize” is any variety of maizethat does not naturally exist in nature. A “non-naturally occurringvariety of maize” can be produced by any method known in the art,including, but not limited to, transforming a maize plant or germplasm,transfecting a maize plant or germplasm and crossing a naturallyoccurring variety of maize with a non-naturally occurring variety ofmaize. In some embodiments, a “non-naturally occurring variety of maize”can comprise one of more heterologous nucleotide sequences. In someembodiments, a “non-naturally occurring variety of maize” can compriseone or more non-naturally occurring copies of a naturally occurringnucleotide sequence (i.e., extraneous copies of a gene that naturallyoccurs in maize).

The “non-Stiff Stalk” heterotic group represents a major heterotic groupin the northern U.S. and Canadian corn growing regions. It can also bereferred to as the “Lancaster” or “Lancaster Sure Crop” heterotic group.

As used herein, the terms “nucleotide sequence,” “polynucleotide,”“nucleic acid sequence,” “nucleic acid molecule” and “nucleic acidfragment” refer to a polymer of RNA or DNA that is single- ordouble-stranded, optionally containing synthetic, non-natural and/oraltered nucleotide bases. A “nucleotide” is a monomeric unit from whichDNA or RNA polymers are constructed and consists of a purine orpyrimidine base, a pentose, and a phosphoric acid group. Nucleotides(usually found in their 5′-monophosphate form) are referred to by theirsingle letter designation as follows: “A” for adenylate ordeoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate ordeoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate,“T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines(C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N”for any nucleotide.

As used herein, the term “nucleotide sequence identity” refers to thepresence of identical nucleotides at corresponding positions of twopolynucleotides. Polynucleotides have “identical” sequences if thesequence of nucleotides in the two polynucleotides is the same whenaligned for maximum correspondence (e.g., in a comparison window).Sequence comparison between two or more polynucleotides is generallyperformed by comparing portions of the two sequences over a comparisonwindow to identify and compare local regions of sequence similarity. Thecomparison window is generally from about 20 to 200 contiguousnucleotides. The “percentage of sequence identity” for polynucleotides,such as about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99 or 100percent sequence identity, can be determined by comparing two optimallyaligned sequences over a comparison window, wherein the portion of thepolynucleotide sequence in the comparison window can include additionsor deletions (i.e., gaps) as compared to the reference sequence foroptimal alignment of the two sequences. The percentage is calculated by:(a) determining the number of positions at which the identical nucleicacid base occurs in both sequences; (b) dividing the number of matchedpositions by the total number of positions in the window of comparison;and (c) multiplying the result by 100. Optimal alignment of sequencesfor comparison can also be conducted by computerized implementations ofknown algorithms, or by visual inspection. Readily available sequencecomparison and multiple sequence alignment algorithms are, respectively,the Basic Local Alignment Search Tool (BLAST) and ClustalW programs,both available on the internet. Other suitable programs include, but arenot limited to, GAP, BestFit, Plot Similarity, and FASTA, which are partof the Accelrys GCG Package available from Accelrys, Inc. of San Diego,Calif., United States of America. In some embodiments, a percentage ofsequence identity refers to sequence identity over the full length ofone of the sequences being compared. In some embodiments, a calculationto determine a percentage of sequence identity does not include in thecalculation any nucleotide positions in which either of the comparednucleic acids includes an “N” (i.e., where any nucleotide could bepresent at that position).

As used herein, the term “percent barren” (PB) refers to the percentageof plants in a given area (e.g., plot) with no grain. It is typicallyexpressed in terms of the percentage of plants per plot and can becalculated as:

$\frac{{number}\mspace{14mu}{of}\mspace{14mu}{plants}\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{plot}\mspace{14mu}{with}\mspace{14mu}{no}\mspace{14mu}{grain}}{{total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{plants}\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{plot}} \times 100$

As used herein, the term “percent yield recovery” (PYREC) refers to theeffect an allele and/or combination of alleles has on the yield of aplant grown under drought stress conditions as compared to that of aplant that is genetically identical except insofar as it lacks theallele and/or combination of alleles. PYREC is calculated as:

$1 - {\frac{\begin{matrix}{{{yield}\mspace{14mu}{under}\mspace{14mu}{full}\mspace{14mu}{irrigation}\;( {{w/{{allele}(s)}}\mspace{14mu}{of}\mspace{14mu}{interest}} )} -} \\{{yield}\mspace{14mu}{under}\mspace{14mu}{drought}\mspace{14mu}{conditions}\;( {{w/{{allele}(s)}}\mspace{14mu}{of}\mspace{14mu}{interest}} )}\end{matrix}}{\begin{matrix}{{{yield}\mspace{14mu}{under}\mspace{14mu}{full}\mspace{14mu}{irrigation}\;( {w\text{/}{out}\mspace{14mu}{{allele}(s)}\mspace{14mu}{of}\mspace{14mu}{interest}} )} -} \\{{yield}\mspace{14mu}{under}\mspace{14mu}{drought}\mspace{14mu}{conditions}\;( {w\text{/}{out}\mspace{14mu}{{allele}(s)}\mspace{14mu}{of}\mspace{14mu}{interest}} )}\end{matrix}} \times 100}$By way of example and not limitation, if a control plant yields 200bushels under full irrigation conditions, but yields only 100 bushelsunder drought stress conditions, then its percentage yield loss would becalculated at 50%. If an otherwise genetically identical hybrid thatcontains the allele(s) of interest yields 125 bushels under droughtstress conditions and 200 bushels under full irrigation conditions, thenthe percentage yield loss would be calculated as 37.5% and the PYRECwould be calculated as 25% [1.00−(200−125)/(200−100)×100)].

As used herein, the phrase “Grain Yield—Well Watered” refers to yieldfrom an area that obtained enough irrigation to prevent plants frombeing water stressed during their growth cycle. In some embodiments,this trait is expressed in bushels per acre.

As used herein, the phrase “Yield Reduction—Hybrid” refers to acalculated trait obtained from a hybrid yield trial grown under stressand non-stress conditions. For a given hybrid, it equals:

$\frac{{{non}\text{-}{stress}\mspace{14mu}{yield}} - {{yield}\mspace{14mu}{under}\mspace{14mu}{stress}}}{{non}\text{-}{stressed}\mspace{14mu}{yield}} \times 100.$In some embodiments, this trait is expressed as percent bushels peracre.

As used herein, the phrase “Yield Reduction—Inbred” refers to acalculated trait obtained from an inbred yield trial grown under stressand non-stress conditions. For a given inbred, it equals:

$\frac{{{non}\text{-}{stress}\mspace{14mu}{yield}} - {{yield}\mspace{14mu}{under}\mspace{14mu}{stress}}}{{non}\text{-}{stressed}\mspace{14mu}{yield}} \times 100.$In some embodiments, this trait is expressed as percent bushels peracre.

As used herein, the phrase “Anthesis Silk Interval” (ASI) refers to thedifference (in some embodiments, expressed in days) between when a plantstarts shedding pollen (anthesis) and it starts producing silk (female).Data are collected on a per plot basis for anthesis and silking and thedifference is calculated.

As used herein, the phrase “Percent Barren” refers to a percentage ofplants in a given area (plot) with no grain. It is typically expressedin terms of plants per plot and can be calculated as:

$\frac{{Number}\mspace{14mu}{of}\mspace{14mu}{plants}\mspace{14mu}{with}\mspace{14mu}{no}\mspace{14mu}{grain}\mspace{14mu}{in}\mspace{14mu} a\mspace{14mu}{plot}}{{Total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{plants}\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{plot}} \times 100.$

As used herein, the terms “phenotype,” “phenotypic trait” or “trait”refer to one or more traits of an organism. The phenotype can beobservable to the naked eye, or by any other means of evaluation knownin the art, e.g., microscopy, biochemical analysis, or anelectromechanical assay. In some cases, a phenotype is directlycontrolled by a single gene or genetic locus, i.e., a “single genetrait.” In other cases, a phenotype is the result of several genes. Itis noted that, as used herein, the term “water optimization phenotype”takes into account environmental conditions that might affect wateroptimization such that the water optimization effect is real andreproducible.

As used herein, the term “plant” can refer to a whole plant, any partthereof, or a cell or tissue culture derived from a plant. Thus, theterm “plant” can refer to any of: whole plants, plant components ororgans (e.g., leaves, stems, roots, etc.), plant tissues, seeds and/orplant cells.

A plant cell is a cell of a plant, taken from a plant, or derivedthrough culture from a cell taken from a plant. Thus, the term “plantcell” includes without limitation cells within seeds, suspensioncultures, embryos, meristematic regions, callus tissue, leaves, shoots,gametophytes, sporophytes, pollen, and microspores. The phrase “plantpart” refers to a part of a plant, including single cells and celltissues such as plant cells that are intact in plants, cell clumps, andtissue cultures from which plants can be regenerated. Examples of plantparts include, but are not limited to, single cells and tissues frompollen, ovules, leaves, embryos, roots, root tips, anthers, flowers,fruits, stems, shoots, and seeds; as well as scions, rootstocks,protoplasts, calli, and the like.

As used herein, the term “polymorphism” refers to a variation in thenucleotide sequence at a locus, where said variation is too common to bedue merely to a spontaneous mutation. A polymorphism must have afrequency of at least about 1% in a population. A polymorphism can be asingle nucleotide polymorphism (SNP), or an insertion/deletionpolymorphism, also referred to herein as an “indel.” Additionally, thevariation can be in a transcriptional profile or a methylation pattern.The polymorphic site or sites of a nucleotide sequence can be determinedby comparing the nucleotide sequences at one or more loci in two or moregermplasm entries.

As used herein, the term “population” refers to a geneticallyheterogeneous collection of plants sharing a common genetic derivation.

As used herein, the term “primer” refers to an oligonucleotide which iscapable of annealing to a nucleic acid target (in some embodiments,annealing specifically to a nucleic acid target) allowing a DNApolymerase to attach, thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of a primerextension product is induced (e.g., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH). In some embodiments, a plurality of primers areemployed to amplify Zea mays nucleic acids (e.g., using the polymerasechain reaction; PCR).

As used herein, the term “probe” refers to a nucleic acid (e.g., asingle stranded nucleic acid or a strand of a double stranded or higherorder nucleic acid, or a subsequence thereof) that can form ahydrogen-bonded duplex with a complementary sequence in a target nucleicacid sequence. Typically, a probe is of sufficient length to form astable and sequence-specific duplex molecule with its complement, and assuch can be employed in some embodiments to detect a sequence ofinterest present in a plurality of nucleic acids.

As used herein, the terms “progeny” and “progeny plant” refer to a plantgenerated from a vegetative or sexual reproduction from one or moreparent plants. A progeny plant can be obtained by cloning or selfing asingle parent plant, or by crossing two parental plants. Thus, thephrase “progeny plant” refers to any plant resulting as progeny from avegetative or sexual reproduction from one or more parent plants ordescendants thereof. For instance, a progeny plant can be obtained bycloning or selfing of a parent plant or by crossing two parental plantsand include selfings as well as the F1 or F2 or still furthergenerations. An F1 is a first-generation progeny produced from parentsat least one of which is used for the first time as donor of a trait,while progeny of second generation (F2) or subsequent generations (F3,F4, and the like) are specimens produced from selfings, intercrosses,backcrosses, or other crosses of F1 s, F2s, and the like. An F1 can thusbe (and in some embodiments is) a hybrid resulting from a cross betweentwo true breeding parents (i.e., parents that are true-breeding are eachhomozygous for a trait of interest or an allele thereof), while an F2can be (and in some embodiments is) a progeny resulting fromself-pollination of the F1 hybrids.

As used herein, the phrase “quantitative trait locus” (QTL; quantitativetrait loci—QTLs) refers to a genetic locus (or loci) that control tosome degree a numerically representable trait that, in some embodiments,is continuously distributed. In some embodiments, a QTL comprises awater optimization associated locus. As used herein, the phrase “wateroptimization associated locus” is used herein to refer to a chromosomalregion containing alleles (e.g., in the form of genes or regulatorysequences) associated with the expression of a water optimizationassociated trait. Thus, a locus “associated with” a water optimizationtrait refers to one or more regions located on one or more chromosomesthat includes at least one gene the expression of which influences wateroptimization and/or at least one regulatory region that controls theexpression of one or more genes involved in one or more wateroptimization traits. The loci can be defined by indicating their geneticlocation in the genome of a given Zea mays plant using one or moremolecular genomic markers. One or more markers, in turn, indicate aspecific locus. Distances between loci are usually measured by thefrequency of crossovers between loci on the same chromosome. The fartherapart two loci are, the more likely that a crossover will occur betweenthem. Conversely, if two loci are close together, a crossover is lesslikely to occur between them. Typically, one centiMorgan (cM) is equalto 1 recombination between loci. When a QTL can be indicated by multiplemarkers, the genetic distance between the end-point markers isindicative of the size of the QTL.

As used herein, the phrase “recombination” refers to an exchange of DNAfragments between two DNA molecules or chromatids of paired chromosomes(a “crossover”) over in a region of similar or identical nucleotidesequences. A “recombination event” is herein understood to refer to ameiotic crossover.

As used herein, the term “reference sequence” refers to a definednucleotide sequence used as a basis for nucleotide sequence comparison.The reference sequence for a marker, for example, is obtained bygenotyping a number of lines at the locus or loci of interest, aligningthe nucleotide sequences in a sequence alignment program, and thenobtaining the consensus sequence of the alignment. Hence, a referencesequence identifies the polymorphisms in alleles at a locus. A referencesequence can not be a copy of an actual nucleic acid sequence from anyparticular organism; however, it is useful for designing primers andprobes for actual polymorphisms in the locus or loci.

As used herein, the term “regenerate”, and grammatical variants thereof,refers to the production of a plant from tissue culture.

As used herein, the phrases “selected allele”, “desired allele”, and“allele of interest” are used interchangeably to refer to a nucleic acidsequence that includes a polymorphic allele associated with a desiredtrait. It is noted that a “selected allele”, “desired allele”, and/or“allele of interest” can be associated with either an increase in adesired trait or a decrease in a desired trait, depending on the natureof the phenotype sought to be generated in an introgressed plant.

As used herein, the phrase “single nucleotide polymorphism”, or “SNP”,refers to a polymorphism that constitutes a single base pair differencebetween two nucleotide sequences. As used herein, the term “SNP” alsorefers to differences between two nucleotide sequences that result fromsimple alterations of one sequence in view of the other that occurs at asingle site in the sequence. For example, the term “SNP” is intended torefer not just to sequences that differ in a single nucleotide as aresult of a nucleic acid substitution in one versus the other, but isalso intended to refer to sequences that differ in 1, 2, 3, or morenucleotides as a result of a deletion of 1, 2, 3, or more nucleotides ata single site in one of the sequences versus the other. It would beunderstood that in the case of two sequences that differ from each otheronly by virtue of a deletion of 1, 2, 3, or more nucleotides at a singlesite in one of the sequences versus the other, this same scenario can beconsidered an addition of 1, 2, 3, or more nucleotides at a single sitein one of the sequences versus the other, depending on which of the twosequences is considered the reference sequence. Single site insertionsand/or deletions are thus also considered to be encompassed by the term“SNP”.

The “Stiff Stalk” heterotic group represents a major heterotic group inthe northern U.S. and Canadian corn growing regions. It can also bereferred to as the “Iowa Stiff Stalk Synthetic” or “BSSS” heteroticgroup.

As used herein, the phrase “stringent hybridization conditions” refersto conditions under which a polynucleotide hybridizes to its targetsubsequence, typically in a complex mixture of nucleic acids, but toessentially no other sequences. Stringent conditions aresequence-dependent and can be different under different circumstances.

Longer sequences typically hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, 1993. Generally, stringent conditions are selectedto be about 5-10° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength pH. The Tm is thetemperature (under defined ionic strength, pH, and nucleic acidconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Exemplary stringent conditions are those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides).

Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide. Additional exemplary stringenthybridization conditions include 50% formamide, 5×SSC, and 1% SDSincubating at 42° C.; or SSC, 1% SDS, incubating at 65° C.; with one ormore washes in 0.2×SSC and 0.1% SDS at 65° C. For PCR, a temperature ofabout 36° C. is typical for low stringency amplification, althoughannealing temperatures can vary between about 32° C. and 48° C. (orhigher) depending on primer length. Additional guidelines fordetermining hybridization parameters are provided in numerous references(see e.g., Ausubel et al., 1999).

As used herein, the phrase “TAQMAN® Assay” refers to real-time sequencedetection using PCR based on the TAQMAN® Assay sold by AppliedBiosystems, Inc. of Foster City, Calif., United States of America. Foran identified marker, a TAQMAN® Assay can be developed for applicationin a breeding program.

As used herein, the term “tester” refers to a line used in a testcrosswith one or more other lines wherein the tester and the line(s) testedare genetically dissimilar. A tester can be an isogenic line to thecrossed line.

As used herein, the term “trait” refers to a phenotype of interest, agene that contributes to a phenotype of interest, as well as a nucleicacid sequence associated with a gene that contributes to a phenotype ofinterest. For example, a “water optimization trait” refers to a wateroptimization phenotype as well as a gene that contributes to a wateroptimization phenotype and a nucleic acid sequence (e.g., an SNP orother marker) that is associated with a water optimization phenotype.

As used herein, the term “transgene” refers to a nucleic acid moleculeintroduced into an organism or its ancestors by some form of artificialtransfer technique. The artificial transfer technique thus creates a“transgenic organism” or a “transgenic cell”. It is understood that theartificial transfer technique can occur in an ancestor organism (or acell therein and/or that can develop into the ancestor organism) and yetany progeny individual that has the artificially transferred nucleicacid molecule or a fragment thereof is still considered transgenic evenif one or more natural and/or assisted breedings result in theartificially transferred nucleic acid molecule being present in theprogeny individual.

An “unfavorable allele” of a marker is a marker allele that segregateswith the unfavorable plant phenotype, therefore providing the benefit ofidentifying plants that can be removed from a breeding program orplanting.

As used herein, the term “water optimization” refers to any measure of aplant, its parts, or its structure that can be measured and/orquantitated in order to assess an extent of or a rate of plant growthand development under conditions of sufficient water availability ascompared to conditions of suboptimal water availability (e.g., drought).As such, a “water optimization trait” is any trait that can be shown toinfluence yield in a plant under different sets of growth conditionsrelated to water availability.

Similarly, “water optimization” can be considered a “phenotype”, whichas used herein refers to a detectable, observable, and/or measurablecharacteristic of a cell or organism. In some embodiments, a phenotypeis based at least in part on the genetic make up of the cell or theorganism (referred to herein as the cell or the organism's “genotype”).Exemplary water optimization phenotypes are grain yield at standardmoisture percentage (YGSMN), grain moisture at harvest (GMSTP), grainweight per plot (GWTPN), and percent yield recovery (PYREC). It is notedthat as used herein, the term “phenotype” takes into account how theenvironment (e.g., environmental conditions) might affect wateroptimization such that the water optimization effect is real andreproducible. As used herein, the term “yield reduction” (YD) refers tothe degree to which yield is reduced in plants grown under stressconditions. YD is calculated as:

$\frac{\begin{matrix}{{{yield}\mspace{14mu}{under}\mspace{14mu}{non}\text{-}{stress}\mspace{11mu}{conditions}} -} \\{{yield}\mspace{14mu}{under}\mspace{14mu}{stress}\mspace{14mu}{conditions}}\end{matrix}}{{yield}\mspace{14mu}{under}\mspace{14mu}{non}\text{-}{stressed}\mspace{14mu}{conditions}} \times 100.$

II. MOLECULAR MARKERS, WATER OPTIMIZATION ASSOCIATED LOCI, ANDCOMPOSITIONS FOR ASSAYING NUCLEIC ACID SEQUENCES

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization can be due to DNA-DNAhybridization techniques after digestion with a restriction enzyme(e.g., an RFLP) and/or due to techniques using the polymerase chainreaction (e.g., STS, SSR/microsatellites, AFLP, and the like). In someembodiments, all differences between two parental genotypes segregate ina mapping population based on the cross of these parental genotypes. Thesegregation of the different markers can be compared and recombinationfrequencies can be calculated. Methods for mapping markers in plants aredisclosed in, for example, Glick & Thompson, 1993; Zietkiewicz et al.,1994. The recombination frequencies of molecular markers on differentchromosomes are generally 50%. Between molecular markers located on thesame chromosome, the recombination frequency generally depends on thedistance between the markers. A low recombination frequency typicallycorresponds to a small genetic distance between markers on a chromosome.Comparing all recombination frequencies results in the most logicalorder of the molecular markers on the chromosomes. This most logicalorder can be depicted in a linkage map (Paterson, 1996). A group ofadjacent or contiguous markers on the linkage map that is associatedwith increased water optimization can provide the position of an MTLassociated with increased water optimization. Genetic loci correlatingwith particular phenotypes, such as drought tolerance, can be mapped inan organism's genome. By identifying a marker or cluster of markers thatco-segregate with a trait of interest, the breeder is able to rapidlyselect a desired phenotype by selecting for the proper marker (a processcalled marker-assisted selection, or MAS). Such markers can also be usedby breeders to design genotypes in silico and to practice whole genomeselection.

The presently disclosed subject matter provides in some embodimentsmarkers associated with enhanced drought tolerance/water optimization.Detection of these markers and/or other linked markers can be used toidentify, select and/or produce drought tolerant plants and/or toeliminate plants that are not drought tolerant from breeding programs orplanting.

The presently disclosed subject matter provides markers associated withimproved water optimization traits. A marker of the presently disclosedsubject matter can comprise a single allele or a combination of allelesat one or more genetic loci. In some embodiments, the one or morealleles are characterized by one or more loci selected from, but notlimited to, the loci represented by SEQ ID NOs: 1-117, 400, and 401,which are located in the Zea mays genome as follows:

(i) SEQ ID NO: 1 is derived from the Zea mays ZmIga4 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 1 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 118 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 119; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a single nucleic polymorphism at nucleotide positions115, 270, 301, and 483 and comprises any part of a DNA sequence within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 1 on Zeamays chromosome 8 that confers an improved water optimization-associatedtrait;

(ii) SEQ ID NO: 2 is derived from a water optimization locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 2 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 120 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 121; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a single nucleic polymorphism at nucleotide positions100 and 264-271 and comprises any part of a DNA sequence within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 2 on Zea mayschromosome 8 that confers an improved water optimization-associatedtrait;

(iii) SEQ ID NO: 3 is derived from the Zea mays ZmDr1 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 2 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 122 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 123; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a single nucleic polymorphism at nucleotide position216 of SEQ ID NO: 3 and comprises any part of a DNA sequence within 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 3 in a Zeamays genome that confers an improved water optimization-associatedtrait;

(iv) SEQ ID NO: 4 is derived from the Zea mays ZmDrA locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 4 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 124 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 125; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a single nucleic polymorphism at nucleotide position503 of SEQ ID NO: 4 and comprises any part of a DNA sequence within 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 4 on Zea mayschromosome 7 that confers an improved water optimization-associatedtrait;

(v) SEQ ID NO: 5 is derived from the Zea mays ZmDr2 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 4 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 126 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 127; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide positions 818-821 of SEQID NO: 5 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 5 on Zea mays chromosome2 that confers an improved water optimization-associated trait;

(vi) SEQ ID NO: 6 is derived from the Zea mays ZmDr3 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 6 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 128 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 129; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 254 of SEQ ID NO:6 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 6 on Zea mays chromosome 2 thatconfers an improved water optimization-associated trait;

(vii) SEQ ID NO: 7 is derived from the Zea mays ZmDr4 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 7 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 130 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 131; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide positions 4497-4498, 4505,4609, 4641, 4792, 4836, 4844, 4969, and 4979-4981 of SEQ ID NO: 7 andcomprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or 25 cM of SEQ ID NO: 7 on Zea mays chromosome 8 thatconfers an improved water optimization-associated trait;

(viii) SEQ ID NO: 8 is derived from the Zea mays ZmMa3 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 8 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 132 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 133; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by polymorphisms at nucleotide positions 217, 390, and 477of SEQ ID NO: 8 and comprises any part of a DNA sequence within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 8 on Zea mayschromosome 2 that confers an improved water optimization-associatedtrait;

(ix) SEQ ID NO: 9 is derived from the Zea mays genome, and is defined bya first oligonucleotide and a second oligonucleotide, wherein saidoligonucleotides can be employed to amplify a subsequence of SEQ ID NO:9 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 134 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 135; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 292 of SEQ ID NO:9 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 9 on Zea mays chromosome 4 thatconfers an improved water optimization-associated trait;

(x) SEQ ID NO: 10 is derived from the Zea mays ZmBglcn locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 10 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 136 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 137; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 166 of SEQ ID NO:10 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 10 on Zea mays chromosome 3that confers an improved water optimization-associated trait;

(xi) SEQ ID NO: 11 is derived from the Zea mays ZmLOC100276591 locus,and is defined by a first oligonucleotide and a second oligonucleotide,wherein said oligonucleotides can be employed to amplify a subsequenceof SEQ ID NO: 11 generated by amplifying a Zea mays nucleic acid with afirst oligonucleotide comprising a nucleotide sequence as set forth inSEQ ID NO: 138 and a second oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 139; and further wherein this locuscomprises alleles of a water optimization-associated trait wherein thealleles are characterized by a polymorphism at nucleotide position 148of SEQ ID NO: 11 and comprises any part of a DNA sequence within 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 11 in the Zeamays genome that confers an improved water optimization-associatedtrait;

(xii) SEQ ID NO: 12 is derived from the Zea mays ZmDr7 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 12 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 140 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 141; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 94 of SEQ ID NO:12 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 12 on Zea mays chromosome 1that confers an improved water optimization-associated trait;

(xiii) SEQ ID NO: 13 is derived from the Zea mays ZmDr7 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 13 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 140 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 141; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by one or more polymorphisms at nucleotide positions 35,86, and/or 89 of SEQ ID NO: 13 and comprises any part of a DNA sequencewithin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 13on Zea mays chromosome 1 that confers an improved wateroptimization-associated trait;

(xiv) SEQ ID NO: 14 is derived from the Zea mays ZmDr8 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 14 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 142 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 143; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 432 of SEQ ID NO:14 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 14 in the Zea mays genome thatconfers an improved water optimization-associated trait;

(xv) SEQ ID NO: 15 is derived from the Zea mays ZmHsp70 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 15 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 144 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 145; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 753 of SEQ ID NO:15 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 15 on Zea mays chromosome 1that confers an improved water optimization-associated trait;

(xvi) SEQ ID NO: 16 is derived from the Zea mays ZmDr9 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 16 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 146 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 147; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 755 of SEQ ID NO:16 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 16 on Zea mays chromosome 4that confers an improved water optimization-associated trait;

(xvii) SEQ ID NO: 17 is derived from the Zea mays ZmDrB locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 17 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 148 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 149; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 431 of SEQ ID NO:17 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 17 in the Zea mays genome thatconfers an improved water optimization-associated trait;

(xviii) SEQ ID NO: 18 is derived from the Zea mays ZmAdh1-1s locus, andis defined by a first oligonucleotide and a second oligonucleotide,wherein said oligonucleotides can be employed to amplify a subsequenceof SEQ ID NO: 18 generated by amplifying a Zea mays nucleic acid with afirst oligonucleotide comprising a nucleotide sequence as set forth inSEQ ID NO: 150 and a second oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 151; and further wherein this locuscomprises alleles of a water optimization-associated trait wherein thealleles are characterized by a polymorphism at nucleotide position 518of SEQ ID NO: 18 and comprises any part of a DNA sequence within 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 18 on Zea mayschromosome 1 that confers an improved water optimization-associatedtrait;

(xix) SEQ ID NO: 19 is derived from the Zea mays ZmDr10 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 19 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 152 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 153; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by one or more polymorphisms at nucleotide positions 182,309, 330, and 463 of SEQ ID NO: 19 and comprises any part of a DNAsequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQID NO: 19 on Zea mays chromosome 8 that confers an improved wateroptimization-associated trait;

(xx) SEQ ID NO: 20 is derived from the Zea mays ZmDrC locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 20 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 154 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 155; and further wherein this locus comprisesone or more alleles of a water optimization-associated trait wherein theone or more alleles are characterized by a polymorphism at nucleotidepositions 773-776 of SEQ ID NO: 20 and comprises any part of a DNAsequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQID NO: 20 in the Zea mays genome that confers an improved wateroptimization-associated trait;

(xxi) SEQ ID NO: 21 is derived from the Zea mays ZmDr5 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 21 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 156 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 157; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by one or more polymorphisms at nucleotide positions 61,200, and 316-324 of SEQ ID NO: 21 and comprises any part of a DNAsequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQID NO: 21 on Zea mays chromosome 5 that confers an improved wateroptimization-associated trait;

(xxii) SEQ ID NO: 22 is derived from the Zea mays ZmDrD locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 22 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 158 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 159; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 211 of SEQ ID NO:22 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 22 on Zea mays chromosome 5that confers an improved water optimization-associated trait;

(xxiii) SEQ ID NO: 23 is derived from a Zea mays water optimizationlocus, and is defined by a first oligonucleotide and a secondoligonucleotide, wherein said oligonucleotides can be employed toamplify a subsequence of SEQ ID NO: 23 generated by amplifying a Zeamays nucleic acid with a first oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 160 and a second oligonucleotidecomprising a nucleotide sequence as set forth in SEQ ID NO: 161; andfurther wherein this locus comprises alleles of a wateroptimization-associated trait wherein the alleles are characterized byone or more polymorphisms at nucleotide positions 116 and 217 of SEQ IDNO: 21 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 23 on Zea mays chromosome 8that confers an improved water optimization-associated trait;

(xxiv) SEQ ID NO: 24 is derived from a Zea mays water optimizationlocus, and is defined by a first oligonucleotide and a secondoligonucleotide, wherein said oligonucleotides can be employed toamplify a subsequence of SEQ ID NO: 24 generated by amplifying a Zeamays nucleic acid with a first oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 162 and a second oligonucleotidecomprising a nucleotide sequence as set forth in SEQ ID NO: 163; andfurther wherein this locus comprises alleles of a wateroptimization-associated trait wherein the alleles are characterized by apolymorphism at nucleotide position 746 of SEQ ID NO: 24 and comprisesany part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or 25 cM of SEQ ID NO: 24 on Zea mays chromosome 8 that confers animproved water optimization-associated trait;

(xxv) SEQ ID NO: 25 is derived from the Zea mays ZmDr12 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 25 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 164 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 165; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 562 of SEQ ID NO:25 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 25 on Zea mays chromosome 8that confers an improved water optimization-associated trait;

(xxvi) SEQ ID NO: 26 is derived from a Zea mays water optimizationlocus, and is defined by a first oligonucleotide and a secondoligonucleotide, wherein said oligonucleotides can be employed toamplify a subsequence of SEQ ID NO: 26 generated by amplifying a Zeamays nucleic acid with a first oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 166 and a second oligonucleotidecomprising a nucleotide sequence as set forth in SEQ ID NO: 167; andfurther wherein this locus comprises alleles of a wateroptimization-associated trait wherein the alleles are characterized by apolymorphism at nucleotide position 1271 of SEQ ID NO: 26 and comprisesany part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or 25 cM of SEQ ID NO: 26 on Zea mays chromosome 8 that confers animproved water optimization-associated trait;

(xxvii) SEQ ID NO: 27 is derived from the Zea mays ZmDrE locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 27 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 168 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 169; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by one or more polymorphisms at nucleotide positions 64and/or 254 of SEQ ID NO: 27 and comprises any part of a DNA sequencewithin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 27on Zea mays chromosome 8 that confers an improved wateroptimization-associated trait;

(xxviii) SEQ ID NO: 28 is derived from the Zea mays ZmDrF locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 28 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 170 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 171; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by one or more polymorphisms at nucleotide positions 98,147, 224, and/or 496 of SEQ ID NO: 28 and comprises any part of a DNAsequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQID NO: 28 on Zea mays chromosome 9 that confers an improved wateroptimization-associated trait;

(xxix) SEQ ID NO: 29 is derived from a Zea mays water optimizationlocus, and is defined by a first oligonucleotide and a secondoligonucleotide, wherein said oligonucleotides can be employed toamplify a subsequence of SEQ ID NO: 29 generated by amplifying a Zeamays nucleic acid with a first oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 172 and a second oligonucleotidecomprising a nucleotide sequence as set forth in SEQ ID NO: 173; andfurther wherein this locus comprises alleles of a wateroptimization-associated trait wherein the alleles are characterized by apolymorphism at nucleotide position 258 of SEQ ID NO: 29 and comprisesany part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or 25 cM of SEQ ID NO: 29 on Zea mays chromosome 8 that confers animproved water optimization-associated trait;

(xxx) SEQ ID NO: 30 is derived from the Zea mays ZmDhn2 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 30 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 174 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 175; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by one or more polymorphisms at nucleotide positions 259,296, 398, and/or 1057 of SEQ ID NO: 30 and comprises any part of a DNAsequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQID NO: 30 on Zea mays chromosome 4 that confers an improved wateroptimization-associated trait;

(xxxi) SEQ ID NO: 31 is derived from the Zea mays ZmDr16 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 31 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 176 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 177; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 239 of SEQ ID NO:31 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 31 on Zea mays chromosome 8that confers an improved water optimization-associated trait;

(xxxii) SEQ ID NO: 32 is derived from the Zea mays ZmDr17 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 32 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 178 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 179; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 208 of SEQ ID NO:32 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 32 in the Zea mays genome thatconfers an improved water optimization-associated trait;

(xxxiii) SEQ ID NO: 33 is derived from a Zea mays water optimizationlocus, and is defined by a first oligonucleotide and a secondoligonucleotide, wherein said oligonucleotides can be employed toamplify a subsequence of SEQ ID NO: 33 generated by amplifying a Zeamays nucleic acid with a first oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 180 and a second oligonucleotidecomprising a nucleotide sequence as set forth in SEQ ID NO: 181; andfurther wherein this locus comprises alleles of a wateroptimization-associated trait wherein the alleles are characterized by apolymorphism at nucleotide position 391 of SEQ ID NO: 33 and comprisesany part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or 25 cM of SEQ ID NO: 33 on Zea mays chromosome 8 that confers animproved water optimization-associated trait;

(xxxiv) SEQ ID NO: 34 is derived from the Zea mays ZmZCN6 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 34 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 182 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 183; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide positions 144-145, 169,and/or 537 of SEQ ID NO: 34 and comprises any part of a DNA sequencewithin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 34on Zea mays chromosome 4 that confers an improved wateroptimization-associated trait;

(xxxv) SEQ ID NO: 35 is derived from the Zea mays ZmDrG locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 35 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 184 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 185; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 76 of SEQ ID NO:35 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 35 on Zea mays chromosome 5that confers an improved water optimization-associated trait;

(xxxvi) SEQ ID NO: 36 is derived from the Zea mays ZmDhn1 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 36 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 186 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 187; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by one or more polymorphisms at nucleotide positions 500,568, and/or 698 of SEQ ID NO: 36 and comprises any part of a DNAsequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQID NO: 36 on Zea mays chromosome 6 that confers an improved wateroptimization-associated trait;

(xxxvii) SEQ ID NO: 37 is derived from the Zea mays ZmDrH locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 37 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 188 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 189; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by one or more polymorphisms at nucleotide positions 375and/or 386 of SEQ ID NO: 37 and comprises any part of a DNA sequencewithin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 37on Zea mays chromosome 5 that confers an improved wateroptimization-associated trait;

(xxxviii) SEQ ID NO: 38 is derived from the Zea mays ZmDrI locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 38 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 190 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 191; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 309 and/or 342 ofSEQ ID NO: 38 and comprises any part of a DNA sequence within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 38 on Zea mayschromosome 3 that confers an improved water optimization-associatedtrait;

(xxxix) SEQ ID NO: 39 is derived from the Zea mays ZmDrJ locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 39 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 192 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 193; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 445 of SEQ ID NO:39 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 39 on Zea mays chromosome 5that confers an improved water optimization-associated trait;

(xl) SEQ ID NO: 40 is derived from the Zea mays ZmH2B1 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 40 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 194 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 195; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 602 of SEQ ID NO:40 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 40 on Zea mays chromosome 4that confers an improved water optimization-associated trait;

(xli) SEQ ID NO: 41 is derived from the Zea mays ZmDr3 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 41 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 196 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 198; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 190 and/or 580 ofSEQ ID NO: 41 and comprises any part of a DNA sequence within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 41 on Zea mayschromosome 2 that confers an improved water optimization-associatedtrait;

(xlii) SEQ ID NO: 42 is derived from the Zea mays ZmDrK locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 42 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 198 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 199; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide positions 238, 266-267,and 808 of SEQ ID NO: 42 and comprises any part of a DNA sequence within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 42 in theZea mays genome that confers an improved water optimization-associatedtrait;

(xliii) SEQ ID NO: 43 is derived from the Zea mays ZmCat1 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 43 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 200 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 201; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 708 of SEQ ID NO:43 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 43 on Zea mays chromosome 5that confers an improved water optimization-associated trait;

(xliv) SEQ ID NO: 44 is derived from a Zea mays water optimizationlocus, and is defined by a first oligonucleotide and a secondoligonucleotide, wherein said oligonucleotides can be employed toamplify a subsequence of SEQ ID NO: 44 generated by amplifying a Zeamays nucleic acid with a first oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 202 and a second oligonucleotidecomprising a nucleotide sequence as set forth in SEQ ID NO: 203; andfurther wherein this locus comprises alleles of a wateroptimization-associated trait wherein the alleles are characterized by apolymorphism at nucleotide position 266 of SEQ ID NO: 44 and comprisesany part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or 25 cM of SEQ ID NO: 44 on Zea mays chromosome 8 that confers animproved water optimization-associated trait;

(xlv) SEQ ID NO: 45 is derived from a Zea mays water optimization locus,and is defined by a first oligonucleotide and a second oligonucleotide,wherein said oligonucleotides can be employed to amplify a subsequenceof SEQ ID NO: 45 generated by amplifying a Zea mays nucleic acid with afirst oligonucleotide comprising a nucleotide sequence as set forth inSEQ ID NO: 202 and a second oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 203; and further wherein this locuscomprises alleles of a water optimization-associated trait wherein thealleles are characterized by a polymorphism at nucleotide position 475of SEQ ID NO: 45 and comprises any part of a DNA sequence within 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 45 on Zea mayschromosome 8 that confers an improved water optimization-associatedtrait;

(xlvi) SEQ ID NO: 46 is derived from a Zea mays water optimizationlocus, and is defined by a first oligonucleotide and a secondoligonucleotide, wherein said oligonucleotides can be employed toamplify a subsequence of SEQ ID NO: 46 generated by amplifying a Zeamays nucleic acid with a first oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 204 and a second oligonucleotidecomprising a nucleotide sequence as set forth in SEQ ID NO: 205; andfurther wherein this locus comprises alleles of a wateroptimization-associated trait wherein the alleles are characterized by apolymorphism at nucleotide position 386 of SEQ ID NO: 46 and comprisesany part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or 25 cM of SEQ ID NO: 46 on Zea mays chromosome 8 that confers animproved water optimization-associated trait;

(xlvii) SEQ ID NO: 47 is derived from a Zea mays water optimizationlocus, and is defined by a first oligonucleotide and a secondoligonucleotide, wherein said oligonucleotides can be employed toamplify a subsequence of SEQ ID NO: 47 generated by amplifying a Zeamays nucleic acid with a first oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 206 and a second oligonucleotidecomprising a nucleotide sequence as set forth in SEQ ID NO: 207; andfurther wherein this locus comprises alleles of a wateroptimization-associated trait wherein the alleles are characterized by apolymorphism at nucleotide position 87 of SEQ ID NO: 47 and comprisesany part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or 25 cM of SEQ ID NO: 47 on Zea mays chromosome 8 that confers animproved water optimization-associated trait;

(xlviii) SEQ ID NO: 48 is derived from a Zea mays water optimizationlocus, and is defined by a first oligonucleotide and a secondoligonucleotide, wherein said oligonucleotides can be employed toamplify a subsequence of SEQ ID NO: 48 generated by amplifying a Zeamays nucleic acid with a first oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 208 and a second oligonucleotidecomprising a nucleotide sequence as set forth in SEQ ID NO: 209; andfurther wherein this locus comprises alleles of a wateroptimization-associated trait wherein the alleles are characterized by apolymorphism at nucleotide position 472 of SEQ ID NO: 48 and comprisesany part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or 25 cM of SEQ ID NO: 48 on Zea mays chromosome 8 that confers animproved water optimization-associated trait;

(xlix) SEQ ID NO: 49 is derived from the Zea mays ZmRIC1 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 49 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 210 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 211; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by one or more polymorphisms at nucleotide positions 166,224, 650, and/or 892 of SEQ ID NO: 49 and comprises any part of a DNAsequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQID NO: 49 on Zea mays chromosome 8 that confers an improved wateroptimization-associated trait;

(l) SEQ ID NO: 50 is derived from the Zea mays ZmPK4 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 50 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 212 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 213; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 541 of SEQ ID NO:50 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 50 on Zea mays chromosome 8that confers an improved water optimization-associated trait;

(li) SEQ ID NO: 51 is derived from the Zea mays ZmPK4 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 51 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 212 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 213; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 111 of SEQ ID NO:51 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 51 on Zea mays chromosome 8that confers an improved water optimization-associated trait;

(lii) SEQ ID NO: 52 is derived from the Zea mays Zpu1 locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 52 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 214 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 215; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 442 of SEQ ID NO:52 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 52 on Zea mays chromosome 2that confers an improved water optimization-associated trait;

(liii) SEQ ID NO: 53 is derived from the Zea mays ZmDrL locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 53 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 216 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 217; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by one or more polymorphisms at nucleotide positions 83,428, 491 and/or 548 of SEQ ID NO: 53 and comprises any part of a DNAsequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQID NO: 53 on Zea mays chromosome 9 that confers an improved wateroptimization-associated trait;

(liv) SEQ ID NO: 54 is derived from the Zea mays ZmDrM locus, and isdefined by a first oligonucleotide and a second oligonucleotide, whereinsaid oligonucleotides can be employed to amplify a subsequence of SEQ IDNO: 54 generated by amplifying a Zea mays nucleic acid with a firstoligonucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 218 and a second oligonucleotide comprising a nucleotide sequence asset forth in SEQ ID NO: 219; and further wherein this locus comprisesalleles of a water optimization-associated trait wherein the alleles arecharacterized by a polymorphism at nucleotide position 126 of SEQ ID NO:54 and comprises any part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 54 on Zea mays chromosome 7that confers an improved water optimization-associated trait;

(lv) SEQ ID NO: 55 is derived from a Zea mays water optimization locus,and is defined by a first oligonucleotide and a second oligonucleotide,wherein said oligonucleotides can be employed to amplify a subsequenceof SEQ ID NO: 55 generated by amplifying a Zea mays nucleic acid with afirst oligonucleotide comprising a nucleotide sequence as set forth inSEQ ID NO: 220 and a second oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 221; and further wherein this locuscomprises alleles of a water optimization-associated trait wherein thealleles are characterized by a polymorphism at nucleotide position 193of SEQ ID NO: 55 and comprises any part of a DNA sequence within 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 55 on Zea mayschromosome 8 that confers an improved water optimization-associatedtrait;

(lvi) SEQ ID NO: 56 is derived from a Zea mays water optimization locus,and is defined by a first oligonucleotide and a second oligonucleotide,wherein said oligonucleotides can be employed to amplify a subsequenceof SEQ ID NO: 56 generated by amplifying a Zea mays nucleic acid with afirst oligonucleotide comprising a nucleotide sequence as set forth inSEQ ID NO: 222 and a second oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 223; and further wherein this locuscomprises alleles of a water optimization-associated trait wherein thealleles are characterized by one or more polymorphisms at nucleotidepositions 237 and/or 516 of SEQ ID NO: 56 and comprises any part of aDNA sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM ofSEQ ID NO: 56 on Zea mays chromosome 8 that confers an improved wateroptimization-associated trait;

(lvii) SEQ ID NO: 57 is derived from a Zea mays water optimizationlocus, and is defined by a first oligonucleotide and a secondoligonucleotide, wherein said oligonucleotides can be employed toamplify a subsequence of SEQ ID NO: 57 generated by amplifying a Zeamays nucleic acid with a first oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 224 and a second oligonucleotidecomprising a nucleotide sequence as set forth in SEQ ID NO: 225; andfurther wherein this locus comprises alleles of a wateroptimization-associated trait wherein the alleles are characterized by apolymorphism at nucleotide position 173 of SEQ ID NO: 57 and comprisesany part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or 25 cM of SEQ ID NO: 57 on Zea mays chromosome 8 that confers animproved water optimization-associated trait;

(lviii) SEQ ID NO: 58 is derived from a Zea mays water optimizationlocus, and is defined by a first oligonucleotide and a secondoligonucleotide, wherein said oligonucleotides can be employed toamplify a subsequence of SEQ ID NO: 58 generated by amplifying a Zeamays nucleic acid with a first oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 226 and a second oligonucleotidecomprising a nucleotide sequence as set forth in SEQ ID NO: 227; andfurther wherein this locus comprises alleles of a wateroptimization-associated trait wherein the alleles are characterized by apolymorphism at nucleotide position 486 of SEQ ID NO: 58 and comprisesany part of a DNA sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or 25 cM of SEQ ID NO: 58 on Zea mays chromosome 8 that confers animproved water optimization-associated trait;

(lix) SEQ ID NO: 59 is derived from a Zea mays water optimization locus,and is defined by a first oligonucleotide and a second oligonucleotide,wherein said oligonucleotides can be employed to amplify a subsequenceof SEQ ID NO: 59 generated by amplifying a Zea mays nucleic acid with afirst oligonucleotide comprising a nucleotide sequence as set forth inSEQ ID NO: 228 and a second oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 229; and further wherein this locuscomprises alleles of a water optimization-associated trait wherein thealleles are characterized by a polymorphism at nucleotide position 729of SEQ ID NO: 59 and comprises any part of a DNA sequence within 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 59 on Zea mayschromosome 8 that confers an improved water optimization-associatedtrait; and

(lx) SEQ ID NO: 60 is derived from a Zea mays water optimization locus,and is defined by a first oligonucleotide and a second oligonucleotide,wherein said oligonucleotides can be employed to amplify a subsequenceof SEQ ID NO: 60 generated by amplifying a Zea mays nucleic acid with afirst oligonucleotide comprising a nucleotide sequence as set forth inSEQ ID NO: 230 and a second oligonucleotide comprising a nucleotidesequence as set forth in SEQ ID NO: 231; and further wherein this locuscomprises alleles of a water optimization-associated trait wherein thealleles are characterized by a polymorphism at nucleotide position 267of SEQ ID NO: 60 and comprises any part of a DNA sequence within 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 60 on Zea mayschromosome 8 that confers an improved water optimization-associatedtrait; and

In some embodiments, a DNA sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or 25 cM of a marker of the presently disclosed subjectmatter displays a genetic recombination frequency of less than about25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% with themarker of the presently disclosed subject matter. In some embodiments,the germplasm is a Zea mays line or variety.

DNA fragments associated with the presence of a water optimizationassociated trait, alleles, and/or haplotypes including, but not limitedto SEQ ID NOs: 1-117, 400, and 401, are also provided. In someembodiments, the DNA fragments associated with the presence of a wateroptimization associated trait have a predicted length and/or nucleicacid sequence, and detecting a DNA fragment having the predicted lengthand/or the predicted nucleic acid sequence is performed such that theamplified DNA fragment has a length that corresponds (plus or minus afew bases; e.g., a length of one, two or three bases more or less) tothe predicted length. In some embodiments, a DNA fragment is anamplified fragment and the amplified fragment has a predicted lengthand/or nucleic acid sequence as does an amplified fragment produced by asimilar reaction with the same primers with the DNA from the plant inwhich the marker was first detected or the nucleic acid sequence thatcorresponds (i.e., as a nucleotide sequence identity of more than 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%) to the expected sequence as based on thesequence of the marker associated with that water optimizationassociated trait in the plant in which the marker was first detected.Upon a review of the instant disclosure, one of ordinary skill in theart would appreciate that markers that are absent in plants while theywere present in at least one parent plant (so-called trans-markers), canalso be useful in assays for detecting a desired trait in an progenyplant, although testing for the absence of a marker to detect thepresence of a specific trait is not optimal. The detecting of anamplified DNA fragment having the predicted length or the predictednucleic acid sequence can be performed by any of a number of techniques,including but not limited to standard gel electrophoresis techniquesand/or by using automated DNA sequencers. The methods are not describedhere in detail as they are well known to the skilled person.

The primer (in some embodiments an extension primer and in someembodiments an amplification primer) is in some embodiments singlestranded for maximum efficiency in extension and/or amplification. Insome embodiments, the primer is an oligodeoxyribonucleotide. A primer istypically sufficiently long to prime the synthesis of extension and/oramplification products in the presence of the agent for polymerization.The minimum lengths of the primers can depend on many factors, includingbut not limited to temperature and composition (A/T vs. G/C content) ofthe primer.

In the context of an amplification primer, these are typically providedas one or more sets of bidirectional primers that include one or moreforward and one or more reverse primers as commonly used in the art ofDNA amplification such as in PCR amplification, As such, it will beunderstood that the term “primer”, as used herein, can refer to morethan one primer, particularly in the case where there is some ambiguityin the information regarding the terminal sequence(s) of the targetregion to be amplified. Hence, a “primer” can include a collection ofprimer oligonucleotides containing sequences representing the possiblevariations in the sequence or includes nucleotides which allow a typicalbase pairing. Primers can be prepared by any suitable method. Methodsfor preparing oligonucleotides of specific sequence are known in theart, and include, for example, cloning, and restriction of appropriatesequences and direct chemical synthesis. Chemical synthesis methods caninclude, for example, the phospho di- or tri-ester method, thediethylphosphoramidate method and the solid support method disclosed inU.S. Pat. No. 4,458,068.

Primers can be labeled, if desired, by incorporating detectable moietiesby for instance spectroscopic, fluorescence, photochemical, biochemical,immunochemical, or chemical moieties.

Template-dependent extension of an oligonucleotide primer is catalyzedby a polymerizing agent in the presence of adequate amounts of the fourdeoxyribonucleotides triphosphates (dATP, dGTP, dCTP and dTTP; i.e.,dNTPs) or analogues, in a reaction medium that comprises appropriatesalts, metal cations, and a pH buffering system. Suitable polymerizingagents are enzymes known to catalyze primer- and template-dependent DNAsynthesis. Known DNA polymerases include, for example, E. coli DNApolymerase or its Klenow fragment, T4 DNA polymerase, and Taq DNApolymerase, as well as various modified versions thereof. The reactionconditions for catalyzing DNA synthesis with these DNA polymerases areknown in the art. The products of the synthesis are duplex moleculesconsisting of the template strands and the primer extension strands,which include the target sequence. These products, in turn, can serve astemplate for another round of replication. In the second round ofreplication, the primer extension strand of the first cycle is annealedwith its complementary primer; synthesis yields a “short” product whichis bound on both the 5′- and the 3′-ends by primer sequences or theircomplements. Repeated cycles of denaturation, primer annealing, andextension can result in the exponential accumulation of the targetregion defined by the primers. Sufficient cycles are run to achieve thedesired amount of polynucleotide containing the target region of nucleicacid. The desired amount can vary, and is determined by the functionwhich the product polynucleotide is to serve.

The PCR method is well described in handbooks and known to the skilledperson. After amplification by PCR, the target polynucleotides can bedetected by hybridization with a probe polynucleotide which forms astable hybrid with that of the target sequence under stringent tomoderately stringent hybridization and wash conditions. If it isexpected that the probes will be essentially completely complementary(i.e., about 99% or greater) to the target sequence, stringentconditions can be used. If some mismatching is expected, for example ifvariant strains are expected with the result that the probe will not becompletely complementary, the stringency of hybridization can bereduced. In some embodiments, conditions are chosen to rule outnon-specific/adventitious binding. Conditions that affect hybridization,and that select against non-specific binding are known in the art, andare described in, for example, Sambrook & Russell, 2001. Generally,lower salt concentration and higher temperature increase the stringencyof hybridization conditions.

In order to detect the presence of two water optimization associatedalleles on a single chromosome in a plant, chromosome painting methodscan also be used. In such methods at least a first water optimizationassociated allele and at least a second water optimization associatedallele can be detected in the same chromosome by in situ hybridizationor in situ PCR techniques. More conveniently, the fact that two wateroptimization associated alleles are present on a single chromosome canbe confirmed by determining that they are in coupling phase: i.e., thatthe traits show reduced segregation when compared to genes residing onseparate chromosomes.

The water optimization associated alleles identified herein are locatedon a number of different chromosomes or linkage groups and theirlocations can be characterized by a number of otherwise arbitrarymarkers. In the present investigations, single nucleotide polymorphisms(SNPs), were used, although restriction fragment length polymorphism(RFLP) markers, amplified fragment length polymorphism (AFLP) markers,microsatellite markers (e.g., SSRs), insertion mutation markers,sequence-characterized amplified region (SCAR) markers, cleavedamplified polymorphic sequence (CAPS) markers, isozyme markers,microarray-based technologies, TAQMAN® Assays, ILLUMINA® GOLDENGATE®Assay analysis, nucleic acid sequencing technologies, or combinations ofthese markers might also have been used, and indeed can be used.

In general, providing complete sequence information for a wateroptimization associated allele and/or haplotype is unnecessary, as theway in which the water optimization associated allele and/or haplotypeis first detected—through an observed correlation between the presenceof one or more single nucleotide polymorphisms and the presence of aparticular phenotypic trait—allows one to trace among a population ofprogeny plants those plants that have the genetic potential forexhibiting a particular phenotypic trait. By providing a non-limitinglist of markers, the presently disclosed subject matter thus providesfor the effective use of the presently disclosed water optimizationassociated alleles and/or haplotypes in breeding programs. In someembodiments, a marker is specific for a particular line of descent.Thus, a specific trait can be associated with a particular marker.

The markers as disclosed herein not only indicate the location of thewater optimization associated allele, they also correlate with thepresence of the specific phenotypic trait in a plant. It is noted thatsingle nucleotide polymorphisms that indicate where a water optimizationassociated allele is present in the genome is non-limiting. In general,the location of a water optimization associated allele is indicated by aset of single nucleotide polymorphisms that exhibit statisticalcorrelation to the phenotypic trait. Once a marker is found outside asingle nucleotide polymorphism (i.e., one that has a LOD-score below acertain threshold, indicating that the marker is so remote thatrecombination in the region between that marker and the wateroptimization associated allele occurs so frequently that the presence ofthe marker does not correlate in a statistically significant manner tothe presence of the phenotype), the boundaries of the water optimizationassociated allele can be considered set. Thus, it is also possible toindicate the location of the water optimization associated allele byother markers located within that specified region. It is further notedthat a single nucleotide polymorphism can also be used to indicate thepresence of the water optimization associated allele (and thus of thephenotype) in an individual plant, which in some embodiments means thatit can be used in marker-assisted selection (MAS) procedures.

In principle, the number of potentially useful markers can be verylarge. Any marker that is linked to a water optimization associatedallele (e.g., falling within the physically boundaries of the genomicregion spanned by the markers having established LOD scores above acertain threshold thereby indicating that no or very littlerecombination between the marker and the water optimization associatedallele occurs in crosses, as well as any marker in linkagedisequilibrium to the water optimization associated allele, as well asmarkers that represent the actual causal mutations within the wateroptimization associated allele) can be used in the presently disclosedmethods and compositions, and are within the scope of the presentlydisclosed subject matter. This means that the markers identified in theapplication as associated with the water optimization associated allele(e.g., markers that are present in or comprise any of SEQ ID NOs: 1-24)are non-limiting examples of markers suitable for use in the presentlydisclosed methods and compositions. Moreover, when a water optimizationassociated allele, or the specific trait-conferring part thereof, isintrogressed into another genetic background (i.e., into the genome ofanother maize or another plant species), then some markers might nolonger be found in the progeny although the trait is present therein,indicating that such markers are outside the genomic region thatrepresents the specific trait-conferring part of the water optimizationassociated allele in the original parent line only and that the newgenetic background has a different genomic organization. Such markers ofwhich the absence indicates the successful introduction of the geneticelement in the progeny are called “trans markers” and can be equallysuitable with respect to the presently disclosed subject matter.

Upon the identification of a water optimization associated allele and/orhaplotype, the water optimization associated allele and/or haplotypeeffect (e.g., the trait) can for instance be confirmed by assessingtrait in progeny segregating for the water optimization associatedalleles and/or haplotypes under investigation. The assessment of thetrait can suitably be performed by using phenotypic assessment as knownin the art for water optimization traits. For example, (field) trialsunder natural and/or irrigated conditions can be conducted to assess thetraits of hybrid and/or inbred maize.

The markers provided by the presently disclosed subject matter can beused for detecting the presence of one or more water optimization traitalleles and/or haplotypes at loci of the presently disclosed subjectmatter in a suspected water optimization trait introgressed maize plant,and can therefore be used in methods involving marker-assisted breedingand selection of such water optimization trait bearing maize plants. Insome embodiments, detecting the presence of a water optimizationassociated allele and/or haplotype of the presently disclosed subjectmatter is performed with at least one of the markers for a wateroptimization associated allele and/or haplotype as defined herein. Thepresently disclosed subject matter therefore relates in another aspectto a method for detecting the presence of a water optimizationassociated allele and/or haplotype for at least one of the presentlydisclosed water optimization traits, comprising detecting the presenceof a nucleic acid sequence of the water optimization associated alleleand/or haplotype in a trait bearing maize plant, which presence can bedetected by the use of the disclosed markers.

In some embodiments, the detecting comprises determining the nucleotidesequence of a Zea mays nucleic acid associated with a water optimizationassociated trait, allele and/or haplotype. The nucleotide sequence of awater optimization associated allele and/or haplotype of the presentlydisclosed subject matter can for instance be resolved by determining thenucleotide sequence of one or more markers associated with the wateroptimization associated allele and/or haplotype and designing internalprimers for the marker sequences that can then be used to furtherdetermine the sequence of the water optimization associated alleleand/or haplotype outside of the marker sequences.

For example, the nucleotide sequence of the SNP markers disclosed hereincan be obtained by isolating the markers from the electrophoresis gelused in the determination of the presence of the markers in the genomeof a subject plant, and determining the nucleotide sequence of themarkers by, for example, dideoxy chain termination sequencing methods,which are well known in the art. In some embodiments of such methods fordetecting the presence of a water optimization associated allele and/orhaplotype in a trait bearing maize plant, the method can also compriseproviding a oligonucleotide or polynucleotide capable of hybridizingunder stringent hybridization conditions to a nucleic acid sequence of amarker linked to the water optimization associated allele and/orhaplotype, in some embodiments selected from the markers disclosedherein, contacting the oligonucleotide or polynucleotide with digestedgenomic nucleic acid of a trait bearing maize plant, and determining thepresence of specific hybridization of the oligonucleotide orpolynucleotide to the digested genomic nucleic acid. In someembodiments, the method is performed on a nucleic acid sample obtainedfrom the trait-bearing maize plant, although in situ hybridizationmethods can also be employed. Alternatively, one of ordinary skill inthe art can, once the nucleotide sequence of the water optimizationassociated allele and/or haplotype has been determined, design specifichybridization probes or oligonucleotides capable of hybridizing understringent hybridization conditions to the nucleic acid sequence of thewater optimization associated allele and/or haplotype and can use suchhybridization probes in methods for detecting the presence of a wateroptimization associated allele and/or haplotype disclosed herein in atrait bearing maize plant.

In some embodiments, the markers can comprise, consist essentially of,or consist of:

-   -   1) a haplotype comprising an A allele at positions 4979-4981 of        SEQ ID NO: 7, an A allele at position 472 of SEQ ID NO: 48, a G        allele at position 237 of SEQ ID NO: 56, a T allele at position        173 of SEQ ID NO: 57, an A allele at position 391 of SEQ ID NO:        33, a G allele at position 116 of SEQ ID NO: 23, a G allele at        position 100 of SEQ ID NO: 2 and a G allele at position 267 of        SEQ ID NO: 60;    -   2) a haplotype comprising a C allele at position 386 of SEQ ID        NO: 46, an A allele at positions 4979-4981 of SEQ ID NO: 7, an A        allele at position 472 of SEQ ID NO: 48, an A allele at position        266 of SEQ ID NO: 44, a T allele at position 309 of SEQ ID NO:        19, a G allele at position 111 of SEQ ID NO: 51, a G allele at        position 562 of SEQ ID NO: 25 and a C allele at position 1271 of        SEQ ID NO: 26;    -   3) a haplotype comprising a G allele at position 87 of SEQ ID        NO: 47, an A allele at position 4641 of SEQ ID NO: 7, a G allele        at position 472 of SEQ ID NO: 48, an A allele at position 266 of        SEQ ID NO: 44, a C allele at position 746 of SEQ ID NO: 24, a C        allele at position 258 of SEQ ID NO: 29, an A allele at position        217 of SEQ ID NO: 23, a G allele at position 100 of SEQ ID NO:        2, a C allele at position 486 of SEQ ID NO: 58 and a G allele at        position 193 of SEQ ID NO: 55;    -   4) a haplotype comprising an A allele at position 4641 of SEQ ID        NO: 7, a G allele and position 237 of SEQ ID NO: 56, an A allele        at position 391 of SEQ ID NO: 33, a T allele at position 309 of        SEQ ID NO: 19, a deletion at positions 264-271 of SEQ ID NO: 2        and a C allele at position 486 of SEQ ID NO: 58;    -   5) a haplotype comprising an A allele at positions 4979-4981 of        SEQ ID NO: 7, a C allele at position 516 of SEQ ID NO: 56, a T        allele at position 475 of SEQ ID NO: 45, an A allele at position        391 of SEQ ID NO: 33, a G allele at position 463 of SEQ ID NO:        19, a G allele at position 254 of SEQ ID NO: 27, a G allele at        position 729 of SEQ ID NO: 59, a G allele at position 267 of SEQ        ID NO: 60 and a G allele at position 193 of SEQ ID NO: 55; or    -   6) a haplotype comprising an A allele at position 4641 of SEQ ID        NO: 7, a G allele at position 237 of SEQ ID NO: 56, a C allele        at position 258 of SEQ ID NO: 29, a G allele at position 463 of        SEQ ID NO: 19 and a G allele at position 193 of SEQ ID NO: 55.

In some embodiments, the marker can comprise, consist essentially of, orconsist of:

-   -   1) a haplotype comprising an A allele at positions 4979-4981 of        SEQ ID NO: 7, an A allele at position 472 of SEQ ID NO: 48 and a        T allele at position 173 of SEQ ID NO: 57;    -   2) a haplotype comprising a C allele at position 386 of SEQ ID        NO: 46, an A allele at positions 4979-4981 of SEQ ID NO: 7, an A        allele at position 472 of SEQ ID NO: 48, an A allele at position        266 of SEQ ID NO: 44, a T allele at position 309 of SEQ ID NO:        19 and a G allele at;    -   3) a haplotype comprising a G allele at position 87 of SEQ ID        NO: 47, an A allele at position 4641 of SEQ ID NO: 7, a G allele        at position 472 of SEQ ID NO: 48, an A allele at position 266 of        SEQ ID NO: 44, a C allele at position 258 of SEQ ID NO: 29 and a        G allele at position 193 of SEQ ID NO: 55;    -   4) a haplotype comprising an A allele at position 4641 of SEQ ID        NO: 7, and a T allele at position 309 of SEQ ID NO: 19;    -   5) a haplotype comprising an A allele at positions 4979-4981 of        SEQ ID NO: 7, a T allele at position 475 of SEQ ID NO: 45, a G        allele at position 463 of SEQ ID NO: 19, and a G allele at        position 193 of SEQ ID NO: 55; or    -   6) a haplotype comprising an A allele at position 4641 of SEQ ID        NO: 7, a C allele at position 258 of SEQ ID NO: 29, a G allele        at position 463 of SEQ ID NO: 19 and a G allele at position 193        of SEQ ID NO: 55.

In some embodiments, the marker can comprise, consist essentially of, orconsist of:

-   -   1) an A allele at positions 4979-4981 of SEQ ID NO: 7;    -   2) an A allele at position 4641 of SEQ ID NO: 7;    -   3) a haplotype comprising a C allele at position 386 of SEQ ID        NO: 46 and an A allele at positions 4979-4981 of SEQ ID NO: 7;        or    -   4) a haplotype comprising an A allele at position 4641 of SEQ ID        NO: 7 and a G allele at position 472 of SEQ ID NO: 48.

In some embodiments, the marker can comprise, consist essentially of, orconsist of:

-   -   1) a haplotype comprising SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,        SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:20 and SEQ        ID NO:25;    -   2) a haplotype comprising SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5,        SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26 and SEQ ID        NO:27;    -   3) a haplotype comprising SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6,        SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID        NO:20, SEQ ID NO:21 and SEQ ID NO:28;    -   4) a haplotype comprising SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:9,        SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:21;    -   5) a haplotype comprising SEQ ID NO:3, SEQ ID NO:8, SEQ ID        NO:10, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:24,        SEQ ID NO:25 and SEQ ID NO:28; or    -   6) a haplotype comprising SEQ ID NO:4, SEQ ID NO:7, SEQ ID        NO:14, SEQ ID NO:17 and SEQ ID NO:28.

In some embodiments, the marker can comprise, consist essentially of, orconsist of:

-   -   1) a haplotype comprising SEQ ID NO:3, SEQ ID NO:5 and SEQ ID        NO:11    -   2) a haplotype comprising SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5,        SEQ ID NO:9, SEQ ID NO:18 and SEQ ID NO:22;    -   3) a haplotype comprising SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6,        SEQ ID NO:9, SEQ ID NO:14 and SEQ ID NO:28;    -   4) a haplotype comprising SEQ ID NO:4, SEQ ID NO:9 and SEQ ID        NO:18;    -   5) a haplotype comprising SEQ ID NO:3, SEQ ID NO:10, SEQ ID        NO:17 and SEQ ID NO:28; or    -   6) a haplotype comprising SEQ ID NO:4, SEQ ID NO:14, SEQ ID        NO:17 and SEQ ID NO:28.

In some embodiments, the marker can comprise, consist essentially of, orconsist of:

-   -   1) SEQ ID NO:3;    -   2) SEQ ID NO:4;    -   3) a haplotype comprising SEQ ID NO:2 and SEQ ID NO:3; or    -   4) a haplotype comprising SEQ ID NO:4 and SEQ ID NO:6.

In some embodiments, the marker can comprise, consist essentially of, orconsist of:

-   -   1) a haplotype comprising SEQ ID NO:87, SEQ ID NO:89, SEQ ID        NO:91, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:104        and SEQ ID NO:109;    -   2) a haplotype comprising SEQ ID NO:86, SEQ ID NO:87, SEQ ID        NO:89, SEQ ID NO:93, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:110        and SEQ ID NO:111;    -   3) a haplotype comprising SEQ ID NO:85, SEQ ID NO:88, SEQ ID        NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:99,        SEQ ID NO:104, SEQ ID NO:105 and SEQ ID NO:112;    -   4) a haplotype comprising SEQ ID NO:88, SEQ ID NO:91, SEQ ID        NO:93, SEQ ID NO:97, SEQ ID NO:102, SEQ ID NO:103 and SEQ ID        NO:105;    -   5) a haplotype comprising SEQ ID NO:87, SEQ ID NO:92, SEQ ID        NO:94, SEQ ID NO:97, SEQ ID NO:101, SEQ ID NO:107, SEQ ID        NO:108, SEQ ID NO:109 and SEQ ID NO:112; or    -   6) a haplotype comprising SEQ ID NO:88, SEQ ID NO:91, SEQ ID        NO:98, SEQ ID NO:101 and SEQ ID NO:112.

In some embodiments, the marker can comprise, consist essentially of, orconsist of:

-   -   1) a haplotype comprising SEQ ID NO:87, SEQ ID NO:89 and SEQ ID        NO:95;    -   2) a haplotype comprising SEQ ID NO:86, SEQ ID NO:87, SEQ ID        NO:89, SEQ ID NO:93, SEQ ID NO:102 and SEQ ID NO:106;    -   3) a haplotype comprising SEQ ID NO:85, SEQ ID NO:88, SEQ ID        NO:90, SEQ ID NO:93, SEQ ID NO:98 and SEQ ID NO:112;    -   4) a haplotype comprising SEQ ID NO:88, SEQ ID NO:93 and SEQ ID        NO:102;    -   5) a haplotype comprising SEQ ID NO:87, SEQ ID NO:94, SEQ ID        NO:101 and SEQ ID NO:112; or    -   6) a haplotype comprising SEQ ID NO:88, SEQ ID NO:98, SEQ ID        NO:101 and SEQ ID NO:112.

In some embodiments, the marker can comprise, consist essentially of, orconsist of:

-   -   1) SEQ ID NO:87;    -   2) SEQ ID NO:88;    -   3) a haplotype comprising SEQ ID NO:86 and SEQ ID NO:87; or    -   4) a haplotype comprising SEQ ID NO:88 and SEQ ID NO:90.

Particular nucleotides that are present at particular locations in themarkers and nucleic acids disclosed herein can be determined usingstandard molecular biology techniques including, but not limited toamplification of genomic DNA from plants and subsequent sequencing.Additionally, oligonucleotide primers can be designed that would beexpected to specifically hybridize to particular sequences that includethe polymorphisms disclosed herein. For example, oligonucleotides can bedesigned to distinguish between the “A” allele and the “G” allele at anucleotide position that corresponds to position 270 of SEQ ID NO: 1using oligonucleotides comprising, consisting essentially of, orconsisting of SEQ ID NOs: 232 and 233. The relevant difference betweenSEQ ID NOs: 232 and 233 is that the former has a G nucleotide atposition 19 and the latter has an A nucleotide at position 19. Thus, SEQID NO: 232 hybridization conditions can be designed that would permitSEQ ID NO: 232 to specifically hybridize to the “G” allele, if present,but not hybridize to the “A” allele, if present. Thus, hybridizationusing these two primers that differ in only one nucleotide can beemployed to assay for the presence of one or the other allele at anucleotide position that corresponds to position 270 of SEQ ID NO: 1.

In some embodiments, the alleles comprising the marker associated withenhanced drought tolerance are detected using a plurality of probesselected from the group consisting of:

(i) SEQ ID NOs: 348 and 349; SEQ ID NOs: 350 and 351; SEQ ID NOs: 360and 361; SEQ ID NOs: 372 and 373; SEQ ID NOs: 382 and 383; SEQ ID NOs:388 and 389; SEQ ID NOs: 382 and 383; and SEQ ID NOs: 398 and 399;

(ii) SEQ ID NOs: 350 and 251; SEQ ID NOs: 356 and 357; SEQ ID NOs: 364and 365; SEQ ID NOs: 366 and 367; SEQ ID NOs: 374 and 375; SEQ ID NOs:378 and 379; SEQ ID NOs: 382 and 383; and SEQ ID NOs: 384 and 385;

(iii) SEQ ID NOs: 348 and 349; SEQ ID NOs: 352 and 353; SEQ ID NOs: 358and 359; SEQ ID NOs: 362 and 363; SEQ ID NOs: 370 and 371; SEQ ID NOs:374 and 375; SEQ ID NOs: 382 and 383; SEQ ID NOs: 386 and 387; and SEQID NOs: 394 and 395;

(iv) SEQ ID NOs: 346 and 347; SEQ ID NOs: 352 and 353; SEQ ID NOs: 356and 357; SEQ ID NOs: 372 and 373; SEQ ID NOs: 388 and 389; and SEQ IDNOs: 394 and 395;

(v) SEQ ID NOs: 351 and 351; SEQ ID NOs: 354 and 355; SEQ ID NOs: 368and 369; SEQ ID NOs: 372 and 373; SEQ ID NOs: 376 and 377; SEQ ID NOs:386 and 387; SEQ ID NOs: 390 and 391; SEQ ID NOs: 396 and 397; and SEQID NOs: 398 and 399;

(vi) SEQ ID NOs: 352 and 353; SEQ ID NOs: 354 and 355; SEQ ID NOs: 370and 371; SEQ ID NOs: 386 and 387; SEQ ID NOs: 388 and 389;

(vii) SEQ ID NOs: 350 and 351; SEQ ID NOs: 382 and 383; SEQ ID NOs: 388and 389; and SEQ ID NOs: 392 and 393;

(viii) SEQ ID NOs: 350 and 351; SEQ ID NOs: 366 and 367; SEQ ID NOs: 374and 375; SEQ ID NOs: 378 and 379; SEQ ID NOs: 382 and 383; and SEQ IDNOs: 384 and 385;

(ix) SEQ ID NOs: 352 and 353; SEQ ID NOs: 370 and 371; SEQ ID NOs: 380and 381; SEQ ID NOs: 382 and 383; and SEQ ID NOs: 386 and 387;

(x) SEQ ID NOs: 352 and 353; SEQ ID NOs: 356 and 357; and SEQ ID NOs:388 and 389;

(xi) SEQ ID NOs: 350 and 351; SEQ ID NOs: 354 and 355; SEQ ID NOs: 376and 377; and SEQ ID NOs: 386 and 387;

(xii) SEQ ID NOs: 350 and 351;

(xiii) SEQ ID NOs: 352 and 353;

(xiv) SEQ ID NOs: 350 and 351 and SEQ ID NOs: 378 and 379; and

(xv) SEQ ID NOs: 352 and 353 and SEQ ID NOs: 382 and 383,

In some embodiments, the marker can comprise, consist essentially of, orconsist of the reverse complement of any of the aforementioned markers.In some embodiments, one or more of the alleles that make up a markerhaplotype is present as described above, whilst one or more of the otheralleles that make up the marker haplotype is present as the reversecomplement of the allele(s) described above. In some embodiments, eachof the alleles that make up a marker haplotype is present as the reversecomplement of the allele(s) described above.

In some embodiments, the marker can comprise, consist essentially of, orconsist of an informative fragment of any of the aforementioned markers,the reverse complement of any of the aforementioned markers, or aninformative fragment of the reverse complement of any of theaforementioned markers. In some embodiments, one or more of thealleles/sequences that make up a marker haplotype is present asdescribed above, whilst one or more of the other alleles/sequences thatmake up the marker haplotype is present as the reverse complement of thealleles/sequences described above. In some embodiments, one or more ofthe alleles/sequences that make up a marker haplotype is present asdescribed above, whilst one or more of the other alleles/sequences thatmake up the marker haplotype is present as an informative fragment ofthe alleles/sequences described above. In some embodiments, one or moreof the alleles/sequences that make up a marker haplotype is present asdescribed above, whilst one or more of the other alleles/sequences thatmake up the marker haplotype is present as an informative fragment ofthe reverse complement of the alleles/sequences described above. In someembodiments, each of the alleles/sequences that make up a markerhaplotype is present as an informative fragment of the alleles/sequencesdescribed above, the reverse complement of the alleles/sequencesdescribed above, or an informative fragment of the reverse complement ofthe alleles/sequences described above.

In some embodiments, the marker can comprise, consist essentially of, orconsist of any marker linked to the aforementioned markers. That is, anyallele and/or haplotype that is in linkage disequilibrium with any ofthe aforementioned markers can also be used to identify, select and/orproduce a maize plant with enhanced drought tolerance. Linked markerscan be determined, for example, by using resources available on theMaizeGDB website.

Isolated and purified markers associated with enhanced drought toleranceare also provided. Such markers can comprise, consist essentially of, orconsist of a nucleotide sequence as set forth in any of SEQ ID NOs:1-117, 400, AND 401, the reverse complement thereof, or an informativefragment thereof. In some embodiments, the marker comprises a detectablemoiety. In some embodiments, the marker permits the detection of one ormore of the marker alleles identified herein.

Compositions comprising a primer pair capable of amplifying a nucleicacid sample isolated from a maize plant or germplasm to generate amarker associated with enhanced drought tolerance are also provided. Insome embodiments, the marker comprises a nucleotide sequence as setforth herein, the reverse complement thereof, or an informative fragmentthereof. In some embodiments, the marker comprises a nucleotide sequencethat is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 99% or 100%, identical to a nucleotide sequence set forth herein,the reverse complement thereof, or an informative fragment thereof. Insome embodiments, the primer pair is one of the amplification primerpairs identified in Table 1 above. One of ordinary skill in the art willunderstand how to select alternative primer pairs according to methodswell known in the art.

Table 5 provides a summary of favorable alleles and single-locushaplotypes that are associated with water optimization.

TABLE 5 Summary of Exemplary Alleles and Single-Locus Haplotypes HAPLSEQ ID NO: Position Favorable* HAPL** CODE 1 115 A (Y) G A 270 A 301 T483 A 2 100 G 264-271 DEL 3 216 G (Y) 4 503 A (Y) 5 818-821 INS (Y) 6254 G (Y); A (P) 7 4497-4498 GA (Y) DEL B 4505 A (Y); A G 4609 C (Y); T(Y) T 4641 T (Y) A 4792 A (Y); G (Y) T 4836 T 4844 C 4969 G 4979-4981TCC 8 217 A (P) A C 390 G 477 A 9 292 C (Y); C (P) 10 166 A (Y); A (P)11 148 G (P) 12 94 C (Y); C 13 35 A (P) 86 C (Y); C 89 G (Y) 14 432 G(P) 15 753 A (Y) 16 755 G (Y) 17 431 G (Y) 18 518 G (P); T (W) 19 182 A(P) G D 309 A (Y); A A 330 G; C (P) G 463 G 20 773-776 C (Y); C (P) 2161 T (Y) C E 200 C 316-324 DEL 22 211 G (P) 23 116 G 217 A 24 746 C 25562 G (Y); G (P); G 26 1271 C 27 64 G F 254 C; T (Y); C (P) T 28 98 T(Y) C G 147 T 224 C 496 T 29 258 C 30 259 T (R) T H 296 G (Y) T 398 A1057 C 31 239 G (Y); G (P) 32 208 G (Y) 33 391 A 34 144-145 C (Y); C (P)169 T (Y) 537 A (P) 35 76 G (Y) 36 500 T (Y) C I 568 G 698 T 37 375 A386 A (P); G (Y); A (Y) 38 309 C (P) 342 A (P) 39 445 G (Y); C (P) 40602 A (Y) 41 190 G (Y); A (P) 580 C (Y) 42 238 INS (Y) A J 266-268 DEL808 C 43 708 C (P); A (Y) 44 266 A 45 475 T 46 386 C 47 87 G 48 472 A; G49 166 G (Y); G (P) C K 224 A 650 G 892 G 50 541 T (Y); A (Y) 51 111 G52 442 C; G 53 83 C (P); T (Y) C L 428 C (Y); C (P) C 491 C 548 C 54 126A (Y) 55 193 G 56 237 G 516 C 57 173 T 58 486 C 59 729 G 60 267 G 400 83C C M 119 A A 601 T T *(Y): YGSMN; (P): GMSTP; (W): GWTPN **HAPL refersto single-locus haplotypes (i.e., haplotypes that comprise a particulargrouping of favorable alleles present together).

As used herein, the phrase “haplotype code” refers to a collection ofSNPs present in a plant, wherein each favorable allele listed in thesingle locus haplotype column of Table 5 is present in the plant. Forexample, a plant is said to have “haplotype A” when at least one locuscorresponding to SEQ ID NO: 1 in the plant has a G at nucleotideposition 115, an A at position 270 of SEQ ID NO: 1, a T at nucleotideposition 301, and an A at position 483. Haplotype A can be alternativelyreferred to as “GATA” with respect to these particular nucleotidepositions. Similarly, a plant is said to have “haplotype C” when atleast one locus correspond to SEQ ID NO: 8 in the plant has an A atnucleotide position 217, a G at nucleotide position 290, and an A atnucleotide position 477 of SEQ ID NO: 8. Haplotype C can thus bealternatively referred to as “AGA” with respect to these particularnucleotide positions.

The identification of plants with different alleles and/or haplotypes ofinterest can provide starting materials for combining alleles and/orhaplotypes in progeny plants via breeding strategies designed to “stack”the alleles and/or haplotypes. As used herein, the term “stacking”, andgrammatical variants thereof, refers to the intentional accumulation bybreeding (including but not limited to crossing two plants, selfing asingle plant, and/or creating a double haploid from a single plant) offavorable water optimization haplotypes in plants such that a plant'sgenome has at least one additional favorable water optimizationhaplotype than its immediate progenitor(s). Stacking includes in someembodiments conveying one or more water optimization traits, alleles,and/or haplotypes into a progeny maize plant such that the progeny maizeplant includes higher number of water optimization traits, alleles,and/or haplotypes than does either parent from which it was derived. Byway of example and not limitation, if Parent 1 has haplotypes A, B, andC, and Parent 2 has haplotypes D, E, and F, “stacking” refers to theproduction of a plant that has any of A, B, and C, with any combinationof D, E, and F. Particularly, “stacking” refers in some embodiments toproducing a plant that has A, B, and C as well as one or more of D, E,and F, or producing a plant that has D, E, and F as well as one or moreof A, B, and C. In some embodiments, “stacking” refers to the productionof a plant from a bi-parental cross that contains all water optimizationassociated haplotypes possessed by either parent.

In some embodiments, the water optimization trait is GrainYield-Drought, and the favorable haplotype comprises a nucleotidesequence comprising a T at nucleotide position 301, a G at nucleotideposition 115, an A at nucleotide position 483, and an A at nucleotideposition 270 of SEQ ID NO: 1; a TCC trinucleotide at nucleotidepositions 4979-4981, a G at nucleotide position 4969, an A at nucleotideposition 4641, a T at nucleotide position 4609, a deletion of nucleotidepositions 4497-4498, a T at nucleotide position 4792, a T at nucleotideposition 4836, a G at nucleotide position 4505, and a C at nucleotideposition 4844 of SEQ ID NO: 7; an A at nucleotide position 217, a G atnucleotide position 390, and an A at nucleotide position 477 of SEQ IDNO: 8; a G at nucleotide position 463, a G at nucleotide position 330, aG at nucleotide position 182, and an A at nucleotide position 309 of SEQID NO: 19; a G at nucleotide position 64 and an A at nucleotide position254 of SEQ ID NO: 27; a C at nucleotide position 98, a T at nucleotideposition 147, a C at nucleotide position 224, and a T at nucleotideposition 496 of SEQ ID NO: 28; a C at nucleotide position 500, a G atnucleotide position 568, and a T at nucleotide position 698 of SEQ IDNO: 36; a deletion of nucleotide positions 266-267, a C at nucleotideposition 808, and an A at nucleotide position 238 of SEQ ID NO: 42;and/or a C at nucleotide position 166, an A at nucleotide position 224,a G at nucleotide position 650, and a G at nucleotide position 892 ofSEQ ID NO: 49.

In some embodiments, the water optimization trait is Grain Yield—WellWatered, and the favorable haplotype comprises a nucleotide sequencecomprising an A at nucleotide position 217, a G at nucleotide position390, and an A at nucleotide position 477 of SEQ ID NO: 8; a C atnucleotide position 500, a G at nucleotide position 568, and a T atnucleotide position 698 of SEQ ID NO: 36; and/or a C at nucleotideposition 83, a C at nucleotide position 548, a C at nucleotide position491, and a C at nucleotide position 428 of SEQ ID NO: 53.

In some embodiments, the water optimization trait is YieldReduction—Hybrid, and the favorable haplotype comprises a nucleotidesequence comprising a C at nucleotide position 98, a T at nucleotideposition 147, a C at nucleotide position 224, and a T at nucleotideposition 496 of SEQ ID NO: 28.

In some embodiments, the water optimization trait is YieldReduction—Inbred, and the favorable haplotype comprises a nucleotidesequence comprising a TCC trinucleotide at nucleotide positions4979-4981, a G at nucleotide position 4969, an A at nucleotide position4641, a T at nucleotide position 4609, a deletion of nucleotidepositions 4497-4498, a T at nucleotide position 4792, a T at nucleotideposition 4836, a G at nucleotide position 4505, and a C at nucleotideposition 4844 of SEQ ID NO: 7; an A at nucleotide position 217, a G atnucleotide position 390, and an A at nucleotide position 477 of SEQ IDNO: 8; a G at nucleotide position 64 and an A at nucleotide position 254of SEQ ID NO: 27; and/or a C at nucleotide position 83, a C atnucleotide position 548, a C at nucleotide position 491, and a C atnucleotide position 428 of SEQ ID NO: 53.

In some embodiments, the water optimization trait is ASI, and thefavorable haplotype comprises a nucleotide sequence comprising a TCCtrinucleotide at nucleotide positions 4979-4981, a G at nucleotideposition 4969, an A at nucleotide position 4641, a T at nucleotideposition 4609, a deletion of nucleotide positions 4497-4498, a T atnucleotide position 4792, a T at nucleotide position 4836, a G atnucleotide position 4505, and a C at nucleotide position 4844 of SEQ IDNO: 7.

In some embodiments, the water optimization trait is Percent Barren, andthe favorable haplotype comprises a nucleotide sequence comprising a TCCtrinucleotide at nucleotide positions 4979-4981, a G at nucleotideposition 4969, an A at nucleotide position 4641, a T at nucleotideposition 4609, a deletion of nucleotide positions 4497-4498, a T atnucleotide position 4792, a T at nucleotide position 4836, a G atnucleotide position 4505, and a C at nucleotide position 4844 of SEQ IDNO: 7; a G at nucleotide position 463, a G at nucleotide position 330, aG at nucleotide position 182, and an A at nucleotide position 309 of SEQID NO: 19; a C at nucleotide position 61, a C at nucleotide position200, and a deletion of nucleotide positions 316-324 of SEQ ID NO: 21;and/or an A at nucleotide position 398, a T at nucleotide position 296,a T at nucleotide position 259, and a C at nucleotide position 1057 ofSEQ ID NO: 30.

In some embodiments of the presently disclosed subject matter, thegenomes of inbred or hybrid Zea mays plants comprise at least three,four, five, six, seven, eight, or nine of haplotypes A-M, whereinhaplotypes A-M are associated with water optimization and are definedherein. In some embodiments, the inbred or hybrid Zea mays plantcomprises a genome comprising Haplotypes C, D, and G; Haplotypes C, D,and L; Haplotypes C, G, and H; Haplotypes C, G, and I; Haplotypes C, I,and L; Haplotypes E, G, and I; Haplotypes F, G, and H; Haplotypes A, C,F, and G; Haplotypes C, E, H, and I; Haplotypes C, G, H, and I;Haplotypes C, H, I, and K; Haplotypes C, H, I, and L; Haplotypes E, F,G, and H; Haplotypes A, C, G, H, and I; Haplotypes B, C, D, G, and L;Haplotypes C, E, G, H, and I; Haplotypes C, G, H, I, and L; HaplotypesA, C, G, H, I, and K; Haplotypes C, E, F, G, H, I, J, K, and L;Haplotypes C, D, G, and M; Haplotypes C, D, L, and M; Haplotypes C, G,H, and M; Haplotypes C, G, I, and M; Haplotypes C, I, L, and M;Haplotypes E, G, I, and M; Haplotypes F, G, H, and M; Haplotypes A, C,F, G, and M; Haplotypes C, E, H, I, and M; Haplotypes C, G, H, I, and M;Haplotypes C, H, I, K, and M; Haplotypes C, H, I, L, and M; HaplotypesE, F, G, H, and M; Haplotypes A, C, G, H, I, and M; Haplotypes B, C, D,G, L, and M; Haplotypes C, E, G, H, I, and M; Haplotypes C, G, H, I, L,and M; Haplotypes A, C, G, H, I, K, and M; and Haplotypes C, E, F, G, H,I, J, K, L, and M. In some embodiments, the inbred or hybrid Zea maysplant is a hybrid plant that is homozygous for at least one ofHaplotypes A-M.

In some embodiments, the inbred or hybrid Zea mays plant comprises agenome comprising Haplotypes A, C, E, G, H, and I, optionally furthercomprising Haplotype M; Haplotypes B, C, D, E, F, G, H, I, and L,optionally further comprising Haplotype M; Haplotypes C, D, E, F, G, H,and L, optionally further comprising Haplotype M; Haplotypes B, C, D, G,I, and L, optionally further comprising Haplotype M; Haplotypes B, C, D,E, G, H, I, and L, optionally further comprising Haplotype M; HaplotypesC, D, E, F, G, H, I, J, K, and L, optionally further comprisingHaplotype M; Haplotypes A, C, G, H, and I, optionally further comprisingHaplotype M; Haplotypes C, E, F, G, H, and I, optionally furthercomprising Haplotype M; Haplotypes C, E, F, G, H, I, and L, optionallyfurther comprising Haplotype M; Haplotypes C, D, E, F, G, and H,optionally further comprising Haplotype M; Haplotypes D, E, F, G, and H,optionally further comprising Haplotype M; Haplotypes A, C, G, H, and I,optionally further comprising Haplotype M; Haplotypes A, C, E, G, H, I,and K, optionally further comprising Haplotype M; Haplotype C, E, G, H,I, and L, optionally further comprising Haplotype M; Haplotypes C, D, E,G, H, I, and L, optionally further comprising Haplotype M; Haplotypes B,C, D, E, G, H, I, and L, optionally further comprising Haplotype M;Haplotypes A, C, G, H, and I, optionally further comprising Haplotype M;Haplotypes A, C, G, H, I, and K, optionally further comprising HaplotypeM; Haplotypes C, G H, I, and L, optionally further comprising HaplotypeM; Haplotypes C, D, G, H, I, and L, optionally further comprisingHaplotype M; Haplotypes B, C, D, G, H, I, and L, optionally furthercomprising Haplotype M; Haplotypes A, C, E, F, G, H, and I, optionallyfurther comprising Haplotype M; Haplotypes A, C, E, F, G, H, I, and K,optionally further comprising Haplotype M; Haplotypes C, E, F, G, H, I,and L, optionally further comprising Haplotype M; Haplotypes C, D, E, F,G, H, I, and L, optionally further comprising Haplotype M; Haplotypes A,C, E, F, G, H, I, J, K, and L, optionally further comprising HaplotypeM; Haplotypes A, C, E, F, G, H, I, J, K, and L, optionally furthercomprising Haplotype M; Haplotypes C, E, F, G, H, I, J, K, and L,optionally further comprising Haplotype M; Haplotypes C, D, E, F, G, H,I, J, K, and L, optionally further comprising Haplotype M; Haplotypes B,C, D, E, F, G, H, I, J, K, and L, optionally further comprisingHaplotype M; Haplotypes A, C, E, F, G, H, and I, optionally furthercomprising Haplotype M; Haplotypes A, C, E, F, G, H, I, and K,optionally further comprising Haplotype M; Haplotypes C, E, F, G, H, I,and L, optionally further comprising Haplotype M; Haplotypes B, C, D, E,F, G, H, and L, optionally further comprising Haplotype M; Haplotypes C,E, F, G, H, I, J, K, and L, optionally further comprising Haplotype M;Haplotypes C, D, G, H, and L, optionally further comprising Haplotype M;Haplotypes C, E, F, G, H, I, and L, optionally further comprisingHaplotype M; and/or Haplotypes B, C, D, E, G, I, and L, optionallyfurther comprising Haplotype M.

As used herein, a plant that comprises multiple haplotypes can also bereferred to by a code designating the haplotypes its posseses. Thus, forexample, a plant that comprises at least one copy of Haplotyes C, D, E,F, G, H, I, J, K, and L in its genome can be referred to as“CDEFGHIJKL”; a plant that comprises at least one copy of Haplotypes B,C, D, E, F, G, H, I, J, K, and L in its genome can be referred to as“BCDEFGHIJKL”, etc. In some embodiments, uppercase and lowercase lettersare employed to further delinate those haplotypes for which a plant (ora cell thereof) is either homozygous (e.g., uppercase) or heterozygous(e.g., lowercase). By way of example and not limitation, a plant or acell that is referred to as CDEFGHIJKL has at least one of Haplotypes C,D, E, F, G, H, I, J, K, and L. In some embodiments, this designationwould indicate that the plant or cell is homozygous for each of thesehaplotypes. Similarly, the designation cdefghijkl indicates that theplant or cell is heterozygous for Haplotypes C, D, E, F, G, H, I, J, K,and L. And finally, the designation CdeFGhijKL indicates that the plantor cell is homozygous for Haplotypes C, F, G, K, and L, is homozygousfor Haplotypes D, E, H, I, and J. In some embodiments, this designationfurther indicates that plant or cell lacks Haplotypes A and B, althoughin some embodiments it indicates that the status of the plant or cellwith respect to these Haplotypes is unknown or untested.

III. METHODS FOR INTROGRESSING ALLELES OF INTEREST AND FOR IDENTIFYINGPLANTS COMPRISING THE SAME

III.A. Marker-Assisted Selection Generally

Markers can be used in a variety of plant breeding applications. Seee.g., Staub et al., Hortscience 31: 729 (1996); Tanksley, PlantMolecular Biology Reporter 1: 3 (1983). One of the main areas ofinterest is to increase the efficiency of backcrossing and introgressinggenes using marker-assisted selection (MAS). In general, MAS takesadvantage of genetic markers that have been identified as having asignificant likelihood of co-segregation with a desired trait. Suchmarkers are presumed to be in/near the gene(s) that give rise to thedesired phenotype, and their presence indicates that the plant willpossess the desired trait. Plants which possess the marker are expectedto transfer the desired phenotype to their progeny.

A marker that demonstrates linkage with a locus affecting a desiredphenotypic trait provides a useful tool for the selection of the traitin a plant population. This is particularly true where the phenotype ishard to assay or occurs at a late stage in plant development. Since DNAmarker assays are less laborious and take up less physical space thanfield phenotyping, much larger populations can be assayed, increasingthe chances of finding a recombinant with the target segment from thedonor line moved to the recipient line. The closer the linkage, the moreuseful the marker, as recombination is less likely to occur between themarker and the gene causing or imparting the trait. Having flankingmarkers decreases the chances that false positive selection will occur.The ideal situation is to have a marker in the gene itself, so thatrecombination cannot occur between the marker and the gene. Such amarker is called a “perfect marker.”

When a gene is introgressed by MAS, it is not only the gene that isintroduced but also the flanking regions. Gepts, Crop Sci 42:1780(2002). This is referred to as “linkage drag.” In the case where thedonor plant is highly unrelated to the recipient plant, these flankingregions carry additional genes that can code for agronomicallyundesirable traits. This “linkage drag” can also result in reduced yieldor other negative agronomic characteristics even after multiple cyclesof backcrossing into the elite maize line. This is also sometimesreferred to as “yield drag.” The size of the flanking region can bedecreased by additional backcrossing, although this is not alwayssuccessful, as breeders do not have control over the size of the regionor the recombination breakpoints. Young et al., Genetics 120:579 (1998).In classical breeding, it is usually only by chance that recombinationswhich contribute to a reduction in the size of the donor segment areselected. Tanksley et al., Biotechnology 7: 257 (1989). Even after 20backcrosses, one can expect to find a sizeable piece of the donorchromosome still linked to the gene being selected. With markers,however, it is possible to select those rare individuals that haveexperienced recombination near the gene of interest. In 150 backcrossplants, there is a 95% chance that at least one plant will haveexperienced a crossover within 1 cM of the gene, based on a singlemeiosis map distance. Markers allow for unequivocal identification ofthose individuals. With one additional backcross of 300 plants, therewould be a 95% chance of a crossover within 1 cM single meiosis mapdistance of the other side of the gene, generating a segment around thetarget gene of less than 2 cM based on a single meiosis map distance.This can be accomplished in two generations with markers, while it wouldhave required on average 100 generations without markers. See Tanksleyet al., supra. When the exact location of a gene is known, flankingmarkers surrounding the gene can be utilized to select forrecombinations in different population sizes. For example, in smallerpopulation sizes, recombinations can be expected further away from thegene, so more distal flanking markers would be required to detect therecombination.

The availability of integrated linkage maps of the maize genomecontaining increasing densities of public maize markers has facilitatedmaize genetic mapping and MAS. See, e.g. the IBM2 Neighbors maps, whichare available online on the MaizeGDB website.

Of all the molecular marker types, SNPs are the most abundant and havethe potential to provide the highest genetic map resolution.Bhattramakki et al., Plant Molec. Biol. 48:539 (2002). SNPs can beassayed in a so-called “ultra-high-throughput” fashion because they donot require large amounts of nucleic acid and automation of the assay isstraight-forward. SNPs also have the benefit of being relativelylow-cost systems. These three factors together make SNPs highlyattractive for use in MAS. Several methods are available for SNPgenotyping, including but not limited to, hybridization, primerextension, oligonucleotide ligation, nuclease cleavage, minisequencingand coded spheres. Such methods have been reviewed in variouspublications: Gut, Hum. Mutat. 17:475 (2001); Shi, Clin. Chem. 47:164(2001); Kwok, Pharmacogenomics 1:95 (2000); Bhattramakki and Rafalski,Discovery and application of single nucleotide polymorphism markers inplants, in PLANT GENOTYPING: THE DNA FINGERPRINTING OF PLANTS, CABIPublishing, Wallingford (2001). A wide range of commercially availabletechnologies utilize these and other methods to interrogate SNPs,including Masscode™ (Qiagen, Germantown, Md.), Invader® (Hologic,Madison, Wis.), SnapShot® (Applied Biosystems, Foster City, Calif.),Taqman® (Applied Biosystems, Foster City, Calif.) and Beadarrays™(Illumina, San Diego, Calif.).

A number of SNPs together within a sequence, or across linked sequences,can be used to describe a haplotype for any particular genotype. Chinget al., BMC Genet. 3:19 (2002); Gupta et al., (2001), Rafalski, PlantSci. 162:329 (2002b). Haplotypes can be more informative than singleSNPs and can be more descriptive of any particular genotype. Forexample, a single SNP can be allele “T” for a specific drought tolerantline or variety, but the allele “T” might also occur in the maizebreeding population being utilized for recurrent parents. In this case,a combination of alleles at linked SNPs can be more informative. Once aunique haplotype has been assigned to a donor chromosomal region, thathaplotype can be used in that population or any subset thereof todetermine whether an individual has a particular gene. The use ofautomated high throughput marker detection platforms known to those ofordinary skill in the art makes this process highly efficient andeffective.

The markers of the presently disclosed subject matter can be used inmarker-assisted selection protocols to identify and/or select progenywith enhanced drought tolerance. Such methods can comprise, consistessentially of, or consist of crossing a first maize plant or germplasmwith a second maize plant or germplasm, wherein the first maize plant orgermplasm comprises a marker associated with enhanced drought tolerance,and selecting a progeny plant that possesses the marker. Either of thefirst and second maize plants, or both, can be of a non-naturallyoccurring variety of maize. In some embodiments, the first maize plantor germplasm is CML333, CML322, Cateto SP VII, Confite Morocho AYA 38,or Tuxpeno VEN 692. In some embodiments, the genome of the first maizeplant or germplasm is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 99% or 100% identical to that of CML333, CML322,Cateto SP VII, Confite Morocho AYA 38, or Tuxpeno VEN 692. In someembodiments, the second maize plant or germplasm is of an elite varietyof maize. In some embodiments, the genome of the second maize plant orgermplasm is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 99% or 100% identical to that of an elite variety of maize. Insome embodiments, the second maize plant is of the NP2391 variety. Insome embodiments, the genome of the second maize plant is at least about50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%identical to that of NP2391.

III.B. Methods of Introgressing Alleles and/or Haplotypes of Interest

Thus, in some embodiments the presently disclosed subject matterprovides methods for introgressing an allele associated with enhanceddrought tolerance into a genetic background lacking said allele. In someembodiments, the methods comprise crossing a donor comprising saidallele with a recurrent parent that lacks said allele; and repeatedlybackcrossing progeny comprising said allele with the recurrent parent,wherein said progeny are identified by detecting, in their genomes, thepresence of a haplotype associated with enhanced drought tolerance,wherein said haplotype is selected from the group consisting of:

-   -   a G nucleotide at the position that corresponds to position 100        of SEQ ID NO: 2, an ACT trinucleotide at the position that        corresponds to positions 4979-4981 of SEQ ID NO: 7, a G        nucleotide at the position that corresponds to position 116 of        SEQ ID NO: 23, an A nucleotide at the position that corresponds        to position 391 of SEQ ID NO: 33, an A nucleotide at the        position that corresponds to position 472 of SEQ ID NO: 48, an A        nucleotide at the position that corresponds to position 237 of        SEQ ID NO: 56, a T nucleotide at the position that corresponds        to position 173 of SEQ ID NO: 57, and a G nucleotide at the        position that corresponds to position 267 of SEQ ID NO: 60;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, an A nucleotide at the        position that corresponds to position 309 of SEQ ID NO: 19, a G        nucleotide at the position that corresponds to position 562 of        SEQ ID NO: 25, a C nucleotide at the position that corresponds        to position 1271 of SEQ ID NO: 26, an A nucleotide at the        position that corresponds to position 266 of SEQ ID NO: 44, a C        nucleotide at the position that corresponds to position 386 of        SEQ ID NO: 46, an A nucleotide at the position that corresponds        to position 472 of SEQ ID NO: 48, and a G nucleotide at the        position that corresponds to position 111 of SEQ ID NO: 51;    -   a G nucleotide at the position that corresponds to position 100,        an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, an A nucleotide at the position that        corresponds to position 217 of SEQ ID NO: 23, a C nucleotide at        the position that corresponds to position 746 of SEQ ID NO: 24,        a C nucleotide at the position that corresponds to position 258        of SEQ ID NO: 29, an A nucleotide at the position that        corresponds to position 266 of SEQ ID NO: 44, a G nucleotide at        the position that corresponds to position 472 of SEQ ID NO: 48,        a G nucleotide at the position that corresponds to position 193        of SEQ ID NO: 55, and a C nucleotide at the position that        corresponds to position 486 of SEQ ID NO: 58;    -   a deletion at nucleotide at the position that corresponds to        positions 264-271 of SEQ ID NO: 2, an A nucleotide at the        position that corresponds to position 4641 of SEQ ID NO: 7, an A        nucleotide at the position that corresponds to position 309 of        SEQ ID NO: 19, an A nucleotide at the position that corresponds        to position 391 of SEQ ID NO: 33, a G nucleotide at the position        that corresponds to position 237 of SEQ ID NO: 56, and a C        nucleotide at the position that corresponds to position 486 of        SEQ ID NO: 58;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, a G nucleotide at the        position that corresponds to position 463 of SEQ ID NO: 19, a C        nucleotide at the position that corresponds to position 254 of        SEQ ID NO: 27, an A nucleotide at the position that corresponds        to position 391 of SEQ ID NO: 33, a T nucleotide at the position        that corresponds to position 475 of SEQ ID NO: 45, a G        nucleotide at the position that corresponds to position 193 of        SEQ ID NO: 55, a C nucleotide at the position that corresponds        to position 516 of SEQ ID NO: 56, a G nucleotide at the position        that corresponds to position 729 of SEQ ID NO: 59, and a G        nucleotide at the position that corresponds to position 267 of        SEQ ID NO: 60;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, a G nucleotide at the position that        corresponds to position 463 of SEQ ID NO: 19, a C nucleotide at        the position that corresponds to position 258 of SEQ ID NO: 29,        a G nucleotide at the position that corresponds to position 193        of SEQ ID NO: 55, and a G nucleotide at the position that        corresponds to position 237 of SEQ ID NO: 56;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, an A nucleotide at the        position that corresponds to position 472 of SEQ ID NO: 48, an A        nucleotide at the position that corresponds to position 237 of        SEQ ID NO: 56, and a T nucleotide at the position that        corresponds to position 173 of SEQ ID NO: 57;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, a C nucleotide at the        position that corresponds to position 1271 of SEQ ID NO: 26, an        A nucleotide at the position that corresponds to position 266 of        SEQ ID NO: 44, a C nucleotide at the position that corresponds        to position 386 of SEQ ID NO: 46, an A nucleotide at the        position that corresponds to position 472 of SEQ ID NO: 48, and        a G nucleotide at the position that corresponds to position 111        of SEQ ID NO: 51;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, a C nucleotide at the position that        corresponds to position 258 of SEQ ID NO: 29, a G nucleotide at        the position that corresponds to position 87 of SEQ ID NO: 47, a        G nucleotide at the position that corresponds to position 472 of        SEQ ID NO: 48, and a G nucleotide at the position that        corresponds to position 193 of SEQ ID NO: 55;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, an A nucleotide at the position that        corresponds to position 309 of SEQ ID NO: 19, and a G nucleotide        at the position that corresponds to position 237 of SEQ ID NO:        56;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, a G nucleotide at the        position that corresponds to position 463 of SEQ ID NO: 19, a T        nucleotide at the position that corresponds to position 475 of        SEQ ID NO: 45, and a G nucleotide at the position that        corresponds to position 193 of SEQ ID NO: 55;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7 and a C nucleotide at the        position that corresponds to position 386 of SEQ ID NO: 46; and    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7 and a G nucleotide at the position that        corresponds to position 472 of SEQ ID NO: 48,    -   and combinations thereof,        thereby producing a drought tolerant maize plant or germplasm        comprising said allele associated with enhanced drought        tolerance in the genetic background of the recurrent parent,        thereby introgressing the allele associated with enhanced        drought tolerance into a genetic background lacking said allele.        In some embodiments, the genome of said drought tolerant maize        plant or germplasm comprising said allele associated with        enhanced drought tolerance is at least about 95% identical to        that of the recurrent parent. In some embodiments, either the        donor or the recurrent parent, or both, is of a non-naturally        occurring variety of maize.

In some embodiments of the presently disclosed methods, the genome ofsaid donor is at least 95% identical to that of CML333, CML322, CatetoSP VII, Confite Morocho AYA 38, or Tuxpeno VEN 692. In some embodiments,said donor is selected from the group consisting of CML333, CML322,Cateto SP VII, Confite Morocho AYA 38, and Tuxpeno VEN 692. In someembodiments, the genome of said recurrent parent plant or germplasm isat least 95% identical to that of an elite variety of maize. In someembodiments, said recurrent parent is of an elite variety of maize. Insome embodiments, 23.

III.D. Methods of Stacking Alleles and/or Haplotypes of Interest

The presently disclosed subject matter relates in some embodiments to“stacking” of haplotypes associated with water optimization in order toproduce plants (and parts thereof) that have multiple favorable wateroptimization haplotypes. By way of example and not limitation, thepresently disclosed subject matter relates in some embodiments to theidentification and characterization of Zea mays loci that are eachassociated with one or more water optimization traits. These locicorrespond to SEQ ID NOs: 1-413.

For each of these loci, favorable haplotypes have been identified thatare associated with water optimization traits. These favorablehaplotypes are summarized herein. The presently disclosed subject matterprovides exemplary haplotypes that are associated with increases anddecreases of various water optimization traits as defined herein. Thephrase “favorable haplotype” refers to a haplotype that when presentresults in a quantitatively higher water optimization versus the casewhen an “unfavorable haplotype” is present. It is noted, however, thenin the case where a lower water optimization is desirable, thehaplotypes disclosed herein as “favorable” could be unfavorablehaplotypes. As such, as used herein, “favorable” is employed in thecontext of increased water optimization, and would be reversed in thecontext of decreased water optimization.

III.E. Methods of Identifying Plants Comprising Alleles and/orHaplotypes of Interest

Methods for identifying a drought tolerant maize plant or germplasm cancomprise detecting the presence of a marker associated with enhanceddrought tolerance. The marker can be detected in any sample taken fromthe plant or germplasm, including, but not limited to, the whole plantor germplasm, a portion of said plant or germplasm (e.g., a cell fromsaid plant or germplasm) or a nucleotide sequence from said plant orgermplasm. The maize plant can be of a non-naturally occurring varietyof maize. In some embodiments, the genome of the maize plant orgermplasm is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 99% or 100% identical to that of an elite variety of maize. Insome embodiments, the genome of the maize plant is at least about 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%, identicalto that of NP2391.

In some embodiments, the presently disclosed subject matter providesmethods for introgressing an allele of interest of a locus associatedwith a water optimization trait into Zea mays germplasm. In someembodiments, the methods comprise:

(a) selecting a Zea mays plant that comprises an allele of interest of alocus associated with a water optimization trait, which allele isdefined by at least one marker allele comprising a polymorphic siteidentifiable by PCR amplification of a Zea mays nucleic acid with a pairof oligonucleotides primers selected from among primer pair 1represented by a primer comprising SEQ ID NO: 118 and a primercomprising SEQ ID NO: 119; primer pair 2 represented by a primercomprising SEQ ID NO: 120 and a primer comprising SEQ ID NO: 121; primerpair 3 represented by a primer comprising SEQ ID NO: 122 and a primercomprising SEQ ID NO: 123; primer pair 4 represented by a primercomprising SEQ ID NO: 124 and a primer comprising SEQ ID NO: 125; primerpair 5 represented by a primer comprising SEQ ID NO: 126 and a primercomprising SEQ ID NO: 127; primer pair 6 represented by a primercomprising SEQ ID NO: 128 and a primer comprising SEQ ID NO: 129; primerpair 7 represented by a primer comprising SEQ ID NO: 130 and a primercomprising SEQ ID NO: 131; primer pair 8 represented by a primercomprising SEQ ID NO: 132 and a primer comprising SEQ ID NO: 133; primerpair 9 represented by a primer comprising SEQ ID NO: 134 and a primercomprising SEQ ID NO: 135; primer pair 10 represented by a primercomprising SEQ ID NO: 136 and a primer comprising SEQ ID NO: 137; primerpair 11 represented by a primer comprising SEQ ID NO: 138 and a primercomprising SEQ ID NO: 139; primer pair 12 represented by a primercomprising SEQ ID NO: 140 and a primer comprising SEQ ID NO: 141; primerpair 13 represented by a primer comprising SEQ ID NO: 142 and a primercomprising SEQ ID NO: 143; primer pair 14 represented by a primercomprising SEQ ID NO: 144 and a primer comprising SEQ ID NO: 145; primerpair 15 represented by a primer comprising SEQ ID NO: 146 and a primercomprising SEQ ID NO: 147; primer pair 16 represented by a primercomprising SEQ ID NO: 148 and a primer comprising SEQ ID NO: 149; primerpair 17 represented by a primer comprising SEQ ID NO: 150 and a primercomprising SEQ ID NO: 151; primer pair 18 represented by a primercomprising SEQ ID NO: 152 and a primer comprising SEQ ID NO: 153; primerpair 19 represented by a primer comprising SEQ ID NO: 154 and a primercomprising SEQ ID NO: 155; primer pair 20 represented by a primercomprising SEQ ID NO: 156 and a primer comprising SEQ ID NO: 157; primerpair 21 represented by a primer comprising SEQ ID NO: 158 and a primercomprising SEQ ID NO: 159; primer pair 22 represented by a primercomprising SEQ ID NO: 160 and a primer comprising SEQ ID NO: 161; primerpair 23 represented by a primer comprising SEQ ID NO: 162 and a primercomprising SEQ ID NO: 163; primer pair 24 represented by a primercomprising SEQ ID NO: 164 and a primer comprising SEQ ID NO: 165; primerpair 25 represented by a primer comprising SEQ ID NO: 166 and a primercomprising SEQ ID NO: 167; primer pair 26 represented by a primercomprising SEQ ID NO: 168 and a primer comprising SEQ ID NO: 169; primerpair 27 represented by a primer comprising SEQ ID NO: 170 and a primercomprising SEQ ID NO: 171; primer pair 28 represented by a primercomprising SEQ ID NO: 172 and a primer comprising SEQ ID NO: 173; primerpair 29 represented by a primer comprising SEQ ID NO: 174 and a primercomprising SEQ ID NO: 175; primer pair 30 represented by a primercomprising SEQ ID NO: 176 and a primer comprising SEQ ID NO: 177; primerpair 31 represented by a primer comprising SEQ ID NO: 178 and a primercomprising SEQ ID NO: 179; primer pair 32 represented by a primercomprising SEQ ID NO: 180 and a primer comprising SEQ ID NO: 181; primerpair 33 represented by a primer comprising SEQ ID NO: 182 and a primercomprising SEQ ID NO: 183; primer pair 34 represented by a primercomprising SEQ ID NO: 184 and a primer comprising SEQ ID NO: 185; primerpair 35 represented by a primer comprising SEQ ID NO: 186 and a primercomprising SEQ ID NO: 187; primer pair 36 represented by a primercomprising SEQ ID NO: 188 and a primer comprising SEQ ID NO: 189; primerpair 37 represented by a primer comprising SEQ ID NO: 190 and a primercomprising SEQ ID NO: 191; primer pair 38 represented by a primercomprising SEQ ID NO: 192 and a primer comprising SEQ ID NO: 193; primerpair 39 represented by a primer comprising SEQ ID NO: 194 and a primercomprising SEQ ID NO: 195; primer pair 40 represented by a primercomprising SEQ ID NO: 196 and a primer comprising SEQ ID NO: 197; primerpair 41 represented by a primer comprising SEQ ID NO: 198 and a primercomprising SEQ ID NO: 199; primer pair 42 represented by a primercomprising SEQ ID NO: 200 and a primer comprising SEQ ID NO: 201; primerpair 43 represented by a primer comprising SEQ ID NO: 202 and a primercomprising SEQ ID NO: 203; primer pair 44 represented by a primercomprising SEQ ID NO: 204 and a primer comprising SEQ ID NO: 205; primerpair 45 represented by a primer comprising SEQ ID NO: 206 and a primercomprising SEQ ID NO: 207; primer pair 46 represented by a primercomprising SEQ ID NO: 208 and a primer comprising SEQ ID NO: 209; primerpair 47 represented by a primer comprising SEQ ID NO: 210 and a primercomprising SEQ ID NO: 211; primer pair 48 represented by a primercomprising SEQ ID NO: 212 and a primer comprising SEQ ID NO: 213; primerpair 49 represented by a primer comprising SEQ ID NO: 214 and a primercomprising SEQ ID NO: 215; primer pair 50 represented by a primercomprising SEQ ID NO: 216 and a primer comprising SEQ ID NO: 217; primerpair 51 represented by a primer comprising SEQ ID NO: 218 and a primercomprising SEQ ID NO: 219; primer pair 52 represented by a primercomprising SEQ ID NO: 220 and a primer comprising SEQ ID NO: 221; primerpair 53 represented by a primer comprising SEQ ID NO: 222 and a primercomprising SEQ ID NO: 223; primer pair 54 represented by a primercomprising SEQ ID NO: 224 and a primer comprising SEQ ID NO: 225; primerpair 55 represented by a primer comprising SEQ ID NO: 226 and a primercomprising SEQ ID NO: 227; primer pair 56 represented by a primercomprising SEQ ID NO: 228 and a primer comprising SEQ ID NO: 229; andprimer pair 57 represented by a primer comprising SEQ ID NO: 230 and aprimer comprising SEQ ID NO: 231; and

(b) introgressing the allele of interest into Zea mays germplasm thatlacks the allele. In some embodiments, the allele of interest comprisesone of SEQ ID NOs: 1-117, 400, and 401 or a nucleotide sequence that isat least 85%, 90%, or 95% identical thereto over the full length of theone of SEQ ID NOs: 1-117, 400, and 401. In some embodiments, the alleleof interest is a favorable allele and/or a favorable haplotype thatpositively correlates with a water optimization trait.

In some embodiments, the favorable allele comprises a nucleotidesequence at least 90% identical to one or more of SEQ ID NOs: 1-117,400, and 401, and further comprises one or more of the particularnucleotide and position combinations disclosed herein. By way of exampleand not limitation, in some embodiments the favorable allele comprises anucleotide sequence at least 90% identical to:

SEQ ID NO: 1, and further comprises a G nucleotide at the position thatcorresponds to position 115 of SEQ ID NO: 1, an A nucleotide at theposition that corresponds to position 270 of SEQ ID NO: 1, a Tnucleotide at the position that corresponds to position 301 of SEQ IDNO: 1, an A nucleotide at the position that corresponds to position 483of SEQ ID NO: 1, or any combination thereof;

SEQ ID NO: 2, and further comprises a G nucleotide at the position thatcorresponds to position 100 and a deletion at the position thatcorresponds to positions 264-271 of SEQ ID NO: 2, or a combinationthereof;

SEQ ID NO: 3, and further comprises a G nucleotide at the position thatcorresponds to position 216 of SEQ ID NO: 3;

SEQ ID NO: 4, and further comprises an A nucleotide at the position thatcorresponds to position 503 of SEQ ID NO: 4;

SEQ ID NO: 5, and further comprises a CGCG tetranucleotide at theposition that corresponds to positions 818-821 of SEQ ID NO: 5;

SEQ ID NO: 6, and further comprises a G or an A nucleotide at theposition that corresponds to position 254 of SEQ ID NO: 6;

SEQ ID NO: 7, and further comprises a GA dinucleotide at the positionthat corresponds to positions 4497-4498 of SEQ ID NO: 7, an A nucleotideat the position that corresponds to position 4641 of SEQ ID NO: 7, a Cor a T nucleotide at the position that corresponds to position 4792 ofSEQ ID NO: 7, a T nucleotide at the position that corresponds toposition 4836 of SEQ ID NO: 7, an ACT or a TCC trinucleotide at theposition that corresponds to positions 4979-4981 of SEQ ID NO: 7, or anycombination thereof; or further comprises a deletion at positions4497-4498 of SEQ ID NO: 7, a G nucleotide at the position thatcorresponds to position 4505 of SEQ ID NO: 7, a T nucleotide at theposition that corresponds to position 4609 of SEQ ID NO: 7, an Anucleotide at the position that corresponds to position 4641 of SEQ IDNO: 7, a T nucleotide at the position that corresponds to position 4792of SEQ ID NO: 7, a T nucleotide at the position that corresponds toposition 4836 of SEQ ID NO: 7, a C nucleotide at the position thatcorresponds to position 4844 of SEQ ID NO: 7, a G nucleotide at theposition that corresponds to position 4969 of SEQ ID NO: 7, and a TCCtrinucleotide at the position that corresponds to positions 4979-4981 ofSEQ ID NO: 7;

SEQ ID NO: 8, and further comprises an A nucleotide at the position thatcorresponds to position 217 of SEQ ID NO: 8, and optionally furthercomprises a G nucleotide at the position that corresponds to position390 of SEQ ID NO: 8 and an A nucleotide at the position that correspondsto position 477 of SEQ ID NO: 8, or any combination thereof;

SEQ ID NO: 9, and further comprises a C nucleotide at the position thatcorresponds to position 292 of SEQ ID NO: 9;

SEQ ID NO: 10, and further comprises an A nucleotide at the positionthat corresponds to position 166 of SEQ ID NO: 10;

SEQ ID NO: 11, and further comprises a G nucleotide at the position thatcorresponds to position 148 of SEQ ID NO: 11;

SEQ ID NO: 12, and further comprises a C nucleotide at the position thatcorresponds to position 94 of SEQ ID NO: 12;

SEQ ID NO: 13, and further comprises an A nucleotide at the positionthat corresponds to position 35 of SEQ ID NO: 13, a C nucleotide at theposition that corresponds to position 148 of SEQ ID NO: 13, or a Gnucleotide at the position that corresponds to position 89 of SEQ ID NO:13, or any combination thereof;

SEQ ID NO: 14, and further comprises a G nucleotide at the position thatcorresponds to position 432 of SEQ ID NO: 14;

SEQ ID NO: 15, and further comprises an A nucleotide at the positionthat corresponds to position 753 of SEQ ID NO: 15;

SEQ ID NO: 16, and further comprises a G nucleotide at the position thatcorresponds to position 755 of SEQ ID NO: 16;

SEQ ID NO: 17, and further comprises a G nucleotide at the position thatcorresponds to position 431 of SEQ ID NO: 17;

SEQ ID NO: 18, and further comprises a G or a T nucleotide at theposition that corresponds to position 518 of SEQ ID NO: 18;

SEQ ID NO: 19, and further comprises an A nucleotide at the positionthat corresponds to position 182 of SEQ ID NO: 19, an A nucleotide atthe position that corresponds to position 309 of SEQ ID NO: 19, or a Gor a C nucleotide at the position that corresponds to position 463 ofSEQ ID NO: 19, or any combination thereof; or that further comprises a Gnucleotide at the position that corresponds to position 182 of SEQ IDNO: 19, an A nucleotide at the position that corresponds to position 309of SEQ ID NO: 19, a G nucleotide at the position that corresponds toposition 330 of SEQ ID NO: 19, and a G nucleotide at the position thatcorresponds to position 463 of SEQ ID NO: 19;

SEQ ID NO: 20, and further comprises a CTGG tetranucleotide at theposition that corresponds to positions 773-776 of SEQ ID NO: 20;

SEQ ID NO: 21, and further comprises a deletion of nucleotide at thepositions that correspond to positions 316-324 of SEQ ID NO: 21; or thatfurther comprises a C nucleotide at the position that corresponds toposition 61 of SEQ ID NO: 21, a C nucleotide at the position thatcorresponds to position 200 of SEQ ID NO: 21, and a deletion at thepositions that correspond to positions 316-324 of SEQ ID NO: 21;

SEQ ID NO: 22, and further comprises a G nucleotide at the position thatcorresponds to position 211 of SEQ ID NO: 22;

SEQ ID NO: 23, and further comprises a G nucleotide at the position thatcorresponds to position 116 of SEQ ID NO: 23, an A nucleotide at theposition that corresponds to position 217 of SEQ ID NO: 23, or acombination thereof;

SEQ ID NO: 24, and further comprises a C nucleotide at the position thatcorresponds to position 746 of SEQ ID NO: 24;

SEQ ID NO: 25, and further comprises a G nucleotide at the position thatcorresponds to position 562 of SEQ ID NO: 25;

SEQ ID NO: 26, and further comprises a C nucleotide at the position thatcorresponds to position 1271 of SEQ ID NO: 26;

SEQ ID NO: 27, and further comprises a C or a T nucleotide at theposition that corresponds to position 254 of SEQ ID NO: 27; or thatoptionally further comprises a G nucleotide at the position thatcorresponds to position 64 of SEQ ID NO: 27 and a T nucleotide at theposition that corresponds to position 254 of SEQ ID NO: 27;

SEQ ID NO: 28, and further comprises a T nucleotide at the position thatcorresponds to position 496 of SEQ ID NO: 28; or further comprises a Cnucleotide at the position that corresponds to position 98 of SEQ ID NO:28, a T nucleotide at the position that corresponds to position 147 ofSEQ ID NO: 28, a C nucleotide at the position that corresponds toposition 224 of SEQ ID NO: 28, and a T nucleotide at the position thatcorresponds to position 496 of SEQ ID NO: 28;

SEQ ID NO: 29, and further comprises a C nucleotide at the position thatcorresponds to position 258 of SEQ ID NO: 29;

SEQ ID NO: 30, and further comprises a T nucleotide at the position thatcorresponds to position 259 of SEQ ID NO: 30, a G nucleotide at theposition that corresponds to position 398 of SEQ ID NO: 30, or acombination thereof; or that further comprises a T nucleotide at theposition that corresponds to position 259 of SEQ ID NO: 30, a Tnucleotide at the position that corresponds to position 296 of SEQ IDNO: 30, an A nucleotide at the position that corresponds to position 398of SEQ ID NO: 30, and a C nucleotide at the position that corresponds toposition 1057 of SEQ ID NO: 30;

SEQ ID NO: 31, and further comprises a G nucleotide at the position thatcorresponds to position 239 of SEQ ID NO: 31;

SEQ ID NO: 32, and further comprises a G nucleotide at the position thatcorresponds to position 208 of SEQ ID NO: 32;

SEQ ID NO: 33, and further comprises an A nucleotide at the positionthat corresponds to position 391 of SEQ ID NO: 33;

SEQ ID NO: 34, and further comprises a CA dinucleotide at the positionthat corresponds to positions 144-145 of SEQ ID NO: 34, a T nucleotideat the position that corresponds to position 169 of SEQ ID NO: 34, an Anucleotide at the position that corresponds to position 537 of SEQ IDNO: 34, or any combination thereof;

SEQ ID NO: 35, and further comprises a G nucleotide at the position thatcorresponds to position 76 of SEQ ID NO: 35;

SEQ ID NO: 36, and further comprises a T nucleotide at the position thatcorresponds to position 698 of SEQ ID NO: 36; or that further comprisesa C nucleotide at the position that corresponds to position 500 of SEQID NO: 36, a G nucleotide at the position that corresponds to position568 of SEQ ID NO: 36, and a T nucleotide at the position thatcorresponds to position 698 of SEQ ID NO: 36;

SEQ ID NO: 37, and further comprises an A nucleotide at the positionthat corresponds to position 375 of SEQ ID NO: 37, an A or a Gnucleotide at the position that corresponds to position 386 of SEQ IDNO: 37, or a combination thereof;

SEQ ID NO: 38, and further comprises a C nucleotide at the position thatcorresponds to position 309 of SEQ ID NO: 38, an A nucleotide at theposition that corresponds to position 342 of SEQ ID NO: 38, or acombination thereof;

SEQ ID NO: 39, and further comprises a G or a C nucleotide at theposition that corresponds to position 445 of SEQ ID NO: 39;

SEQ ID NO: 40, and further comprises an A nucleotide at the positionthat corresponds to position 602 of SEQ ID NO: 40;

SEQ ID NO: 41, and further comprises a G or an A nucleotide at theposition that corresponds to position 190 of SEQ ID NO: 41, a Cnucleotide at the position that corresponds to position 580 of SEQ IDNO: 41, or a combination thereof;

SEQ ID NO: 42, and further comprises a TTG trinucleotide at the positionthat corresponds to positions 266-268 of SEQ ID NO: 42; or that furthercomprises an A nucleotide at the position that corresponds to position238 of SEQ ID NO: 42, a deletion of the nucleotides that corresponds topositions 266-268 of SEQ ID NO: 42, and a C nucleotide at the positionthat corresponds to position 808 of SEQ ID NO: 42;

SEQ ID NO: 43, and further comprises a C or an A nucleotide at theposition that corresponds to position 708 of SEQ ID NO: 43;

SEQ ID NO: 44, and further comprises an A nucleotide at the positionthat corresponds to position 266 of SEQ ID NO: 44;

SEQ ID NO: 45, and further comprises a T nucleotide at the position thatcorresponds to position 475 of SEQ ID NO: 45;

SEQ ID NO: 46, and further comprises a C nucleotide at the position thatcorresponds to position 386 of SEQ ID NO: 46;

SEQ ID NO: 47, and further comprises a G nucleotide at the position thatcorresponds to position 87 of SEQ ID NO: 47;

SEQ ID NO: 48, and further comprises an A or a G nucleotide at theposition that corresponds to position 472 of SEQ ID NO: 48;

SEQ ID NO: 49, and further comprises a G nucleotide at the position thatcorresponds to position 650 of SEQ ID NO: 49, and optionally alsofurther comprises a C nucleotide at the position that corresponds toposition 166 of SEQ ID NO: 49, and A nucleotide at the position thatcorresponds to position 224 of SEQ ID NO: 49, and a G nucleotide at theposition that corresponds to position 892 of SEQ ID NO: 49;

SEQ ID NO: 50, and further comprises a T or an A nucleotide at theposition that corresponds to position 541 of SEQ ID NO: 50;

SEQ ID NO: 51, and further comprises a G nucleotide at the position thatcorresponds to position 111 of SEQ ID NO: 51;

SEQ ID NO: 52, and further comprises a C or a G nucleotide at theposition that corresponds to position 442 of SEQ ID NO: 52;

SEQ ID NO: 53, and further comprises a C or a T nucleotide at theposition that corresponds to position 428 of SEQ ID NO: 53, a Cnucleotide at the position that corresponds to position 491 of SEQ IDNO: 53, or a combination thereof; or that further comprises a Cnucleotide at the positions that correspond to at positions 83, 428,491, and 548 of SEQ ID NO: 53;

SEQ ID NO: 54, and further comprises an A nucleotide at the positionthat corresponds to position 126 of SEQ ID NO: 54;

SEQ ID NO: 55, and further comprises a G nucleotide at the position thatcorresponds to position 193 of SEQ ID NO: 55;

SEQ ID NO: 56, and further comprises and A or a G nucleotide at theposition that corresponds to position 237 of SEQ ID NO: 56, a Cnucleotide at the position that corresponds to position 516 of SEQ IDNO: 56, or a combination thereof;

SEQ ID NO: 57, and further comprises a T nucleotide at the position thatcorresponds to position 173 of SEQ ID NO: 57;

SEQ ID NO: 58, and further comprises a C nucleotide at the position thatcorresponds to position 486 of SEQ ID NO: 58;

SEQ ID NO: 59, and further comprises a G nucleotide at the position thatcorresponds to position 729 of SEQ ID NO: 59; and/or SEQ ID NO: 60, andfurther comprises a G nucleotide at the position that corresponds toposition 267 of SEQ ID NO: 60.

Methods for producing a drought tolerant maize plant can comprisedetecting, in a germplasm, a marker associated with enhanced droughttolerance and producing a maize plant from said germplasm. The germplasmcan be of a non-naturally occurring variety of maize. In someembodiments, the genome of the germplasm is at least about 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identical tothat of an elite variety of maize. In some embodiments, the genome ofthe germplasm is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 99% or 100% identical to that of NP2391.

In some embodiments, the alleles comprising the marker associated withenhanced drought tolerance are detected using a plurality of probesselected from the group consisting of:

-   -   1) a haplotype comprising SEQ ID NO:87, SEQ ID NO:89, SEQ ID        NO:91, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:104        and SEQ ID NO:109;    -   2) a haplotype comprising SEQ ID NO:86, SEQ ID NO:87, SEQ ID        NO:89, SEQ ID NO:93, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:110        and SEQ ID NO:111;    -   3) a haplotype comprising SEQ ID NO:85, SEQ ID NO:88, SEQ ID        NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:99,        SEQ ID NO:104, SEQ ID NO:105 and SEQ ID NO:112; 4) a haplotype        comprising SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:93, SEQ ID        NO:97, SEQ ID NO:102, SEQ ID NO:103 and SEQ ID NO:105;    -   5) a haplotype comprising SEQ ID NO:87, SEQ ID NO:92, SEQ ID        NO:94, SEQ ID NO:97, SEQ ID NO:101, SEQ ID NO:107, SEQ ID        NO:108, SEQ ID NO:109 and SEQ ID NO:112; and    -   6) a haplotype comprising SEQ ID NO:88, SEQ ID NO:91, SEQ ID        NO:98, SEQ ID NO:101 and SEQ ID NO:112.

In some embodiments, the alleles comprising the marker associated withenhanced drought tolerance are detected using a plurality of probesselected from the group consisting of:

-   -   1) a haplotype comprising SEQ ID NO:87, SEQ ID NO:89 and SEQ ID        NO:95;    -   2) a haplotype comprising SEQ ID NO:86, SEQ ID NO:87, SEQ ID        NO:89, SEQ ID NO:93, SEQ ID NO:102 and SEQ ID NO:106;    -   3) a haplotype comprising SEQ ID NO:85, SEQ ID NO:88, SEQ ID        NO:90, SEQ ID NO:93, SEQ ID NO:98 and SEQ ID NO:112;    -   4) a haplotype comprising SEQ ID NO:88, SEQ ID NO:93 and SEQ ID        NO:102;    -   5) a haplotype comprising SEQ ID NO:87, SEQ ID NO:94, SEQ ID        NO:101 and SEQ ID NO:112; and    -   6) a haplotype comprising SEQ ID NO:88, SEQ ID NO:98, SEQ ID        NO:101 and SEQ ID NO:112.

In some embodiments, the allele(s) comprising the marker associated withenhanced drought tolerance is/are detected using a probe or probesselected from the group consisting of:

-   -   1) SEQ ID NO:87;    -   2) SEQ ID NO:88;    -   3) a haplotype comprising SEQ ID NO:86 and SEQ ID NO:87; and    -   4) a haplotype comprising SEQ ID NO:88 and SEQ ID NO:90.

In some embodiments, the alleles comprising the marker associated withenhanced drought tolerance are detected in amplification products from anucleic acid sample isolated from a maize plant or germplasm, whereinthe amplification products are produced using pairs of amplificationprimers selected from the group consisting of:

-   -   1) SEQ ID NO:31 and SEQ ID NO:59, SEQ ID NO:33 and SEQ ID NO:61,        SEQ ID NO:35 and SEQ ID NO:63, SEQ ID NO:39 and SEQ ID NO:67,        SEQ ID NO:41 and SEQ ID NO:69, SEQ ID NO:44 and SEQ ID NO:72,        SEQ ID NO:48 and SEQ ID NO:76, and SEQ ID NO:53 and SEQ ID        NO:81;    -   2) SEQ ID NO:30 and SEQ ID NO:58, SEQ ID NO:31 and SEQ ID NO:59,        SEQ ID NO:33 and SEQ ID NO:61, SEQ ID NO:37 and SEQ ID NO:65,        SEQ ID NO:46 and SEQ ID NO:74, SEQ ID NO:50 and SEQ ID NO:78,        SEQ ID NO:54 and SEQ ID NO:82, and SEQ ID NO:55 and SEQ ID        NO:83;    -   3) SEQ ID NO:29 and SEQ ID NO:57, SEQ ID NO:32 and SEQ ID NO:60,        SEQ ID NO:34 and SEQ ID NO:62, SEQ ID NO:37 and SEQ ID NO:65,        SEQ ID NO:40 and SEQ ID NO:68, SEQ ID NO:42 and SEQ ID NO:70,        SEQ ID NO:43 and SEQ ID NO:71, SEQ ID NO:48 and SEQ ID NO:76,        SEQ ID NO:49 and SEQ ID NO:77, and SEQ ID NO:56 and SEQ ID        NO:84;    -   4) SEQ ID NO:32 and SEQ ID NO:60, SEQ ID NO:35 and SEQ ID NO:63,        SEQ ID NO:37 and SEQ ID NO:65, SEQ ID NO:41 and SEQ ID NO:69,        SEQ ID NO:46 and SEQ ID NO:74, SEQ ID NO:47 and SEQ ID NO:75,        and SEQ ID NO:49 and SEQ ID NO:77;    -   5) SEQ ID NO:31 and SEQ ID NO:59, SEQ ID NO:36 and SEQ ID NO:64,        SEQ ID NO:38 and SEQ ID NO:66, SEQ ID NO:41 and SEQ ID NO:69,        SEQ ID NO:45 and SEQ ID NO:73, SEQ ID NO:51 and SEQ ID NO:79,        SEQ ID NO:52 and SEQ ID NO:80, SEQ ID NO:53 and SEQ ID NO:81,        and SEQ ID NO:56 and SEQ ID NO:84; and    -   6) SEQ ID NO:32 and SEQ ID NO:60, SEQ ID NO:35 and SEQ ID NO:63,        SEQ ID NO:42 and SEQ ID NO:70, SEQ ID NO:45 and SEQ ID NO:73,        and SEQ ID NO:56 and SEQ ID NO:84.

In some embodiments, the alleles comprising the marker associated withenhanced drought tolerance are detected in amplification products from anucleic acid sample isolated from a maize plant or germplasm, whereinthe amplification products are produced using pairs of amplificationprimers selected from the group consisting of:

-   -   1) SEQ ID NO:31 and SEQ ID NO:59, SEQ ID NO:33 and SEQ ID NO:61        and SEQ ID NO:39 and SEQ ID NO:67;    -   2) SEQ ID NO:30 and SEQ ID NO:58, SEQ ID NO:31 and SEQ ID NO:59,        SEQ ID NO:33 and SEQ ID NO:61, SEQ ID NO:37 and SEQ ID NO:65,        SEQ ID NO:46 and SEQ ID NO:74, and SEQ ID NO:50 and SEQ ID        NO:78;    -   3) SEQ ID NO:29 and SEQ ID NO:57, SEQ ID NO:32 and SEQ ID NO:60,        SEQ ID NO:34 and SEQ ID NO:62, SEQ ID NO:37 and SEQ ID NO:65,        SEQ ID NO:42 and SEQ ID NO:70, and SEQ ID NO:56 and SEQ ID        NO:84;    -   4) SEQ ID NO:32 and SEQ ID NO:60, SEQ ID NO:37 and SEQ ID NO:65,        and SEQ ID NO:46 and SEQ ID NO:74;    -   5) SEQ ID NO:31 and SEQ ID NO:59, SEQ ID NO:38 and SEQ ID NO:66,        SEQ ID NO:45 and SEQ ID NO:73, and SEQ ID NO:56 and SEQ ID        NO:84; and    -   6) SEQ ID NO:32 and SEQ ID NO:60, SEQ ID NO:42 and SEQ ID NO:70,        SEQ ID NO:45 and SEQ ID NO:73, and SEQ ID NO:56 and SEQ ID        NO:84.

In some embodiments, the allele(s) comprising the marker associated withenhanced drought tolerance is/are detected in an amplification productor products from a nucleic acid sample isolated from a maize plant orgermplasm,

wherein the amplification product(s) is/are produced using a pair (orpairs) of amplification primers selected from the group consisting of:

-   -   1) SEQ ID NO:31 and SEQ ID NO:59;    -   2) SEQ ID NO:32 and SEQ ID NO:60;    -   3) SEQ ID NO:30 and SEQ ID NO:58, and SEQ ID NO:31 and SEQ ID        NO:59; and    -   4) SEQ ID NO:32 and SEQ ID NO:60, and SEQ ID NO:34 and SEQ ID        NO:62.

Methods for introgressing an allele associated with enhanced droughttolerance into a maize plant or germplasm can comprise crossing a firstmaize plant or germplasm comprising said allele (the donor) with asecond maize plant or germplasm that lacks said allele (the recurrentparent) and repeatedly backcrossing progeny comprising said allele withthe recurrent parent. Progeny comprising said allele can be identifiedby detecting, in their genomes, the presence of a marker associated withenhanced drought tolerance. Either the donor or the recurrent parent, orboth, can be of a non-naturally occurring variety of maize. In someembodiments, the donor is CML333, CML322, Cateto SP VII, Confite MorochoAYA 38, or Tuxpeno VEN 692. In some embodiments, the genome of the donoris at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,99% or 100% identical to that of CML333, CML322, Cateto SP VII, ConfiteMorocho AYA 38, or Tuxpeno VEN 692. In some embodiments, the recurrentparent is of an elite variety of maize. In some embodiments, the genomeof the recurrent parent is at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 99% or 100% identical to that of an elitevariety of maize. In some embodiments, the recurrent parent is of theNP2391 variety. In some embodiments, the genome of the recurrent parentis at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,99% or 100% identical to that of NP2391.

In some embodiments, the presently disclosed subject matter alsoprovides methods of producing a drought tolerant maize plant. In someembodiments, the presently disclosed methods comprise detecting, in amaize germplasm, the presence of a marker associated with enhanceddrought tolerance, wherein said marker is selected from the groupconsisting of:

-   -   a G nucleotide at the position that corresponds to position 100        of SEQ ID NO: 2, an ACT trinucleotide at the position that        corresponds to positions 4979-4981 of SEQ ID NO: 7, a G        nucleotide at the position that corresponds to position 116 of        SEQ ID NO: 23, an A nucleotide at the position that corresponds        to position 391 of SEQ ID NO: 33, an A nucleotide at the        position that corresponds to position 472 of SEQ ID NO: 48, an A        nucleotide at the position that corresponds to position 237 of        SEQ ID NO: 56, a T nucleotide at the position that corresponds        to position 173 of SEQ ID NO: 57, and a G nucleotide at the        position that corresponds to position 267 of SEQ ID NO: 60;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, an A nucleotide at the        position that corresponds to position 309 of SEQ ID NO: 19, a G        nucleotide at the position that corresponds to position 562 of        SEQ ID NO: 25, a C nucleotide at the position that corresponds        to position 1271 of SEQ ID NO: 26, an A nucleotide at the        position that corresponds to position 266 of SEQ ID NO: 44, a C        nucleotide at the position that corresponds to position 386 of        SEQ ID NO: 46, an A nucleotide at the position that corresponds        to position 472 of SEQ ID NO: 48, and a G nucleotide at the        position that corresponds to position 111 of SEQ ID NO: 51;    -   a G nucleotide at the position that corresponds to position 100,        an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, an A nucleotide at the position that        corresponds to position 217 of SEQ ID NO: 23, a C nucleotide at        the position that corresponds to position 746 of SEQ ID NO: 24,        a C nucleotide at the position that corresponds to position 258        of SEQ ID NO: 29, an A nucleotide at the position that        corresponds to position 266 of SEQ ID NO: 44, a G nucleotide at        the position that corresponds to position 472 of SEQ ID NO: 48,        a G nucleotide at the position that corresponds to position 193        of SEQ ID NO: 55, and a C nucleotide at the position that        corresponds to position 486 of SEQ ID NO: 58;    -   a deletion at nucleotide at the position that corresponds to        positions 264-271 of SEQ ID NO: 2, an A nucleotide at the        position that corresponds to position 4641 of SEQ ID NO: 7, an A        nucleotide at the position that corresponds to position 309 of        SEQ ID NO: 19, an A nucleotide at the position that corresponds        to position 391 of SEQ ID NO: 33, a G nucleotide at the position        that corresponds to position 237 of SEQ ID NO: 56, and a C        nucleotide at the position that corresponds to position 486 of        SEQ ID NO: 58;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, a G nucleotide at the        position that corresponds to position 463 of SEQ ID NO: 19, a C        nucleotide at the position that corresponds to position 254 of        SEQ ID NO: 27, an A nucleotide at the position that corresponds        to position 391 of SEQ ID NO: 33, a T nucleotide at the position        that corresponds to position 475 of SEQ ID NO: 45, a G        nucleotide at the position that corresponds to position 193 of        SEQ ID NO: 55, a C nucleotide at the position that corresponds        to position 516 of SEQ ID NO: 56, a G nucleotide at the position        that corresponds to position 729 of SEQ ID NO: 59, and a G        nucleotide at the position that corresponds to position 267 of        SEQ ID NO: 60;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, a G nucleotide at the position that        corresponds to position 463 of SEQ ID NO: 19, a C nucleotide at        the position that corresponds to position 258 of SEQ ID NO: 29,        a G nucleotide at the position that corresponds to position 193        of SEQ ID NO: 55, and a G nucleotide at the position that        corresponds to position 237 of SEQ ID NO: 56;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, an A nucleotide at the        position that corresponds to position 472 of SEQ ID NO: 48, an A        nucleotide at the position that corresponds to position 237 of        SEQ ID NO: 56, and a T nucleotide at the position that        corresponds to position 173 of SEQ ID NO: 57;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, a C nucleotide at the        position that corresponds to position 1271 of SEQ ID NO: 26, an        A nucleotide at the position that corresponds to position 266 of        SEQ ID NO: 44, a C nucleotide at the position that corresponds        to position 386 of SEQ ID NO: 46, an A nucleotide at the        position that corresponds to position 472 of SEQ ID NO: 48, and        a G nucleotide at the position that corresponds to position 111        of SEQ ID NO: 51;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, a C nucleotide at the position that        corresponds to position 258 of SEQ ID NO: 29, a G nucleotide at        the position that corresponds to position 87 of SEQ ID NO: 47, a        G nucleotide at the position that corresponds to position 472 of        SEQ ID NO: 48, and a G nucleotide at the position that        corresponds to position 193 of SEQ ID NO: 55;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, an A nucleotide at the position that        corresponds to position 309 of SEQ ID NO: 19, and a G nucleotide        at the position that corresponds to position 237 of SEQ ID NO:        56;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, a G nucleotide at the        position that corresponds to position 463 of SEQ ID NO: 19, a T        nucleotide at the position that corresponds to position 475 of        SEQ ID NO: 45, and a G nucleotide at the position that        corresponds to position 193 of SEQ ID NO: 55;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7 and a C nucleotide at the        position that corresponds to position 386 of SEQ ID NO: 46; and    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7 and a G nucleotide at the position that        corresponds to position 472 of SEQ ID NO: 48,    -   and combinations thereof; and

producing a plant from said maize germplasm, thereby producing a droughttolerant maize plant.

The presently disclosed subject matter also provides methods foridentifying and/or selecting a drought tolerant maize plant orgermplasm. Methods for selecting a drought tolerant maize plant orgermplasm can comprise crossing a first maize plant or germplasm with asecond maize plant or germplasm, wherein said first maize plant orgermplasm comprises a marker associated with enhanced drought tolerance,and selecting a progeny plant or germplasm comprising said markerassociated with enhanced drought tolerance. Either the first or secondmaize plant or germplasm, or both, can be of a non-naturally occurringvariety of maize. In some embodiments, the first maize plant orgermplasm is CML333, CML322, Cateto SP VII, Confite Morocho AYA 38, orTuxpeno VEN 692. In some embodiments, the genome of the first maizeplant or germplasm is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 99% or 100% identical to that of CML333, CML322,Cateto SP VII, Confite Morocho AYA 38, or Tuxpeno VEN 692. In someembodiments, the second maize plant or germplasm is of an elite varietyof maize. In some embodiments, the genome of the second maize plant orgermplasm is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 99% or 100% identical to that of an elite variety of maize. Insome embodiments, the second maize plant or germplasm is of the NP2391variety. In some embodiments, the genome of the second maize plant orgermplasm is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 99% or 100% identical to that of NP2391.

Thus, in some embodiments the methods comprise detecting, in said maizeplant or germplasm, the presence of a marker associated with enhanceddrought tolerance, wherein said marker comprises a plurality of alleles,which are detected in amplification products from a nucleic acid sampleisolated from said maize plant or germplasm, said amplification productshaving been produced using pairs of amplification primers selected fromthe group consisting of:

-   -   (i) SEQ ID NOs: 120 and 121; SEQ ID NOs: 130 and 131; SEQ ID        NOs: 160 and 161; SEQ ID NOs: 180 and 181; SEQ ID NOs: 208 and        209; SEQ ID NOs: 222 and 223; SEQ ID NOs: 224 and 225; and SEQ        ID NOs: 230 and 231;    -   (ii) SEQ ID NOs: 130 and 131; SEQ ID NOs: 152 and 153; SEQ ID        NOs: 164 and 165; SEQ ID NOs: 166 and 167; SEQ ID NOs: 202 and        203; SEQ ID NOs: 204 and 205; SEQ ID NOs: 208 and 209; and SEQ        ID NOs: 212 and 213;    -   (iii) SEQ ID NOs: 120 and 121; SEQ ID NOs: 130 and 131; SEQ ID        NOs: 160 and 161; SEQ ID NOs: 162 and 163; SEQ ID NOs: 172 and        173; SEQ ID NOs: 202 and 203; SEQ ID NOs: 206 and 207; SEQ ID        NOs: 208 and 209; SEQ ID NOs: 220 and 221; and SEQ ID NOs: 226        and 227;    -   (iv) SEQ ID NOs: 120 and 121; SEQ ID NOs: 130 and 131; SEQ ID        NOs: 152 and 153; SEQ ID NOs: 180 and 181; SEQ ID NOs: 202 and        203; SEQ ID NOs: 222 and 223; and SEQ ID NOs: 226 and 227;    -   (v) SEQ ID NOs: 130 and 131; SEQ ID NOs: 152 and 153; SEQ ID        NOs: 168 and 169; SEQ ID NOs: 180 and 181; SEQ ID NOs: 202 and        203; SEQ ID NOs: 220 and 221; SEQ ID NOs: 222 and 223; SEQ ID        NOs: 228 and 229; and SEQ ID NOs: 230 and 231;    -   (vi) SEQ ID NOs: 130 and 131; SEQ ID NOs: 152 and 153; SEQ ID        NOs: 172 and 173; SEQ ID NOs: 220 and 221; and SEQ ID NOs: 222        and 223;    -   (vii) SEQ ID NOs: 130 and 131; SEQ ID NOs: 208 and 209; SEQ ID        NOs: 222 and 223; and SEQ ID NOs: 224 and 225;    -   (viii) SEQ ID NOs: 130 and 131; SEQ ID NOs: 152 and 153; SEQ ID        NOs: 166 and 167; SEQ ID NOs: 202 and 203; SEQ ID NOs: 204 and        205; SEQ ID NOs: 208 and 209; and SEQ ID NOs: 212 and 213,    -   (ix) SEQ ID NOs: 130 and 131; SEQ ID NOs: 172 and 173; SEQ ID        NOs: 202 and 203; SEQ ID NOs: 206 and 207; SEQ ID NOs: 208 and        209; and SEQ ID NOs: 220 and 221;    -   (x) SEQ ID NOs: 130 and 131; SEQ ID NOs: 152 and 153; SEQ ID        NOs: 202 and 203; and SEQ ID NOs: 222 and 223;    -   (xi) SEQ ID NOs: 130 and 131; SEQ ID NOs: 152 and 153; SEQ ID        NOs: 202 and 203; and SEQ ID NOs: 220 and 221;    -   (xii) SEQ ID NOs: 130 and 131;    -   (xiii) SEQ ID NOs: 130 and 131; and SEQ ID NOs: 204 and 205; and    -   (xiv) SEQ ID NOs: 130 and 131; and SEQ ID NOs: 208 and 209,

thereby identifying and/or selecting a drought tolerant maize plant orgermplasm.

The presently disclosed subject matter also provides methods forproducing a drought tolerant maize plant comprising detecting, in amaize germplasm, the presence of a marker associated with enhanceddrought tolerance, wherein said marker comprises a plurality of alleles,which are detected in amplification products from a nucleic acid sampleisolated from said maize plant or germplasm, said amplification producthaving been produced using pairs of amplification primers selected fromthe group consisting of:

-   -   (i) SEQ ID NOs: 120 and 121; SEQ ID NOs: 130 and 131; SEQ ID        NOs: 160 and 161; SEQ ID NOs: 180 and 181; SEQ ID NOs: 208 and        209; SEQ ID NOs: 222 and 223; SEQ ID NOs: 224 and 225; and SEQ        ID NOs: 230 and 231;    -   (ii) SEQ ID NOs: 130 and 131; SEQ ID NOs: 152 and 153; SEQ ID        NOs: 164 and 165; SEQ ID NOs: 166 and 167; SEQ ID NOs: 202 and        203; SEQ ID NOs: 204 and 205; SEQ ID NOs: 208 and 209; and SEQ        ID NOs: 212 and 213;    -   (iii) SEQ ID NOs: 120 and 121; SEQ ID NOs: 130 and 131; SEQ ID        NOs: 160 and 161; SEQ ID NOs: 162 and 163; SEQ ID NOs: 172 and        173; SEQ ID NOs: 202 and 203; SEQ ID NOs: 206 and 207; SEQ ID        NOs: 208 and 209; SEQ ID NOs: 220 and 221; and SEQ ID NOs: 226        and 227;    -   (iv) SEQ ID NOs: 120 and 121; SEQ ID NOs: 130 and 131; SEQ ID        NOs: 152 and 153; SEQ ID NOs: 180 and 181; SEQ ID NOs: 202 and        203; SEQ ID NOs: 222 and 223; and SEQ ID NOs: 226 and 227;    -   (v) SEQ ID NOs: 130 and 131; SEQ ID NOs: 152 and 153; SEQ ID        NOs: 168 and 169; SEQ ID NOs: 180 and 181; SEQ ID NOs: 202 and        203; SEQ ID NOs: 220 and 221; SEQ ID NOs: 222 and 223; SEQ ID        NOs: 228 and 229; and SEQ ID NOs: 230 and 231;    -   (vi) SEQ ID NOs: 130 and 131; SEQ ID NOs: 152 and 153; SEQ ID        NOs: 172 and 173; SEQ ID NOs: 220 and 221; and SEQ ID NOs: 222        and 223;    -   (vii) SEQ ID NOs: 130 and 131; SEQ ID NOs: 208 and 209; SEQ ID        NOs: 222 and 223; and SEQ ID NOs: 224 and 225;    -   (viii) SEQ ID NOs: 130 and 131; SEQ ID NOs: 152 and 153; SEQ ID        NOs: 166 and 167; SEQ ID NOs: 202 and 203; SEQ ID NOs: 204 and        205; SEQ ID NOs: 208 and 209; and SEQ ID NOs: 212 and 213,    -   (ix) SEQ ID NOs: 130 and 131; SEQ ID NOs: 172 and 173; SEQ ID        NOs: 202 and 203; SEQ ID NOs: 206 and 207; SEQ ID NOs: 208 and        209; and SEQ ID NOs: 220 and 221;    -   (x) SEQ ID NOs: 130 and 131; SEQ ID NOs: 152 and 153; SEQ ID        NOs: 202 and 203; and SEQ ID NOs: 222 and 223;    -   (xi) SEQ ID NOs: 130 and 131; SEQ ID NOs: 152 and 153; SEQ ID        NOs: 202 and 203; and SEQ ID NOs: 220 and 221;    -   (xii) SEQ ID NOs: 130 and 131;    -   (xiii) SEQ ID NOs: 130 and 131; and SEQ ID NOs: 204 and 205; and    -   (xiv) SEQ ID NOs: 130 and 131; and SEQ ID NOs: 208 and 209, and        producing a plant from said maize germplasm, thereby producing a        drought tolerant maize plant.

The presently disclosed subject matter also provides methods foridentifying and/or selecting a drought tolerant maize plant orgermplasm, comprising detecting, in said maize plant or germplasm, thepresence of a marker associated with enhanced drought tolerance, whereinsaid marker comprises a plurality of alleles, which are detected using aplurality of probes selected from the group consisting of:

-   -   (i) SEQ ID NOs: 348 and 349; SEQ ID NOs: 350 and 351; SEQ ID        NOs: 360 and 361; SEQ ID NOs: 372 and 373; SEQ ID NOs: 382 and        383; SEQ ID NOs: 388 and 389; SEQ ID NOs: 382 and 383; and SEQ        ID NOs: 398 and 399;    -   (ii) SEQ ID NOs: 350 and 251; SEQ ID NOs: 356 and 357; SEQ ID        NOs: 364 and 365; SEQ ID NOs: 366 and 367; SEQ ID NOs: 374 and        375; SEQ ID NOs: 378 and 379; SEQ ID NOs: 382 and 383; and SEQ        ID NOs: 384 and 385;    -   (iii) SEQ ID NOs: 348 and 349; SEQ ID NOs: 352 and 353; SEQ ID        NOs: 358 and 359; SEQ ID NOs: 362 and 363; SEQ ID NOs: 370 and        371; SEQ ID NOs: 374 and 375; SEQ ID NOs: 382 and 383; SEQ ID        NOs: 386 and 387; and SEQ ID NOs: 394 and 395;    -   (iv) SEQ ID NOs: 346 and 347; SEQ ID NOs: 352 and 353; SEQ ID        NOs: 356 and 357; SEQ ID NOs: 372 and 373; SEQ ID NOs: 388 and        389; and SEQ ID NOs: 394 and 395;    -   (v) SEQ ID NOs: 351 and 351; SEQ ID NOs: 354 and 355; SEQ ID        NOs: 368 and 369; SEQ ID NOs: 372 and 373; SEQ ID NOs: 376 and        377; SEQ ID NOs: 386 and 387; SEQ ID NOs: 390 and 391; SEQ ID        NOs: 396 and 397; and SEQ ID NOs: 398 and 399;    -   (vi) SEQ ID NOs: 352 and 353; SEQ ID NOs: 354 and 355; SEQ ID        NOs: 370 and 371; SEQ ID NOs: 386 and 387; SEQ ID NOs: 388 and        389;    -   (vii) SEQ ID NOs: 350 and 351; SEQ ID NOs: 382 and 383; SEQ ID        NOs: 388 and 389; and SEQ ID NOs: 392 and 393;    -   (viii) SEQ ID NOs: 350 and 351; SEQ ID NOs: 366 and 367; SEQ ID        NOs: 374 and 375; SEQ ID NOs: 378 and 379; SEQ ID NOs: 382 and        383; and SEQ ID NOs: 384 and 385;    -   (ix) SEQ ID NOs: 352 and 353; SEQ ID NOs: 370 and 371; SEQ ID        NOs: 380 and 381; SEQ ID NOs: 382 and 383; and SEQ ID NOs: 386        and 387;    -   (x) SEQ ID NOs: 352 and 353; SEQ ID NOs: 356 and 357; and SEQ ID        NOs: 388 and 389;    -   (xi) SEQ ID NOs: 350 and 351; SEQ ID NOs: 354 and 355; SEQ ID        NOs: 376 and 377; and SEQ ID NOs: 386 and 387;    -   (xii) SEQ ID NOs: 350 and 351;    -   (xiii) SEQ ID NOs: 352 and 353;    -   (xiv) SEQ ID NOs: 350 and 351 and SEQ ID NOs: 378 and 379; and    -   (xv) SEQ ID NOs: 352 and 353 and SEQ ID NOs: 382 and 383,        thereby identifying and/or selecting a drought tolerant maize        plant or germplasm.

The presently disclosed subject matter also provides methods forproducing a drought tolerant maize plant comprising detecting, in amaize germplasm, the presence of a marker associated with enhanceddrought tolerance, wherein said marker comprises a plurality of alleles,which are detected using a plurality of probes selected from the groupconsisting of:

-   -   (i) SEQ ID NOs: 348 and 349; SEQ ID NOs: 350 and 351; SEQ ID        NOs: 360 and 361; SEQ ID NOs: 372 and 373; SEQ ID NOs: 382 and        383; SEQ ID NOs: 388 and 389; SEQ ID NOs: 382 and 383; and SEQ        ID NOs: 398 and 399;    -   (ii) SEQ ID NOs: 350 and 251; SEQ ID NOs: 356 and 357; SEQ ID        NOs: 364 and 365; SEQ ID NOs: 366 and 367; SEQ ID NOs: 374 and        375; SEQ ID NOs: 378 and 379; SEQ ID NOs: 382 and 383; and SEQ        ID NOs: 384 and 385;    -   (iii) SEQ ID NOs: 348 and 349; SEQ ID NOs: 352 and 353; SEQ ID        NOs: 358 and 359; SEQ ID NOs: 362 and 363; SEQ ID NOs: 370 and        371; SEQ ID NOs: 374 and 375; SEQ ID NOs: 382 and 383; SEQ ID        NOs: 386 and 387; and SEQ ID NOs: 394 and 395;    -   (iv) SEQ ID NOs: 346 and 347; SEQ ID NOs: 352 and 353; SEQ ID        NOs: 356 and 357; SEQ ID NOs: 372 and 373; SEQ ID NOs: 388 and        389; and SEQ ID NOs: 394 and 395;    -   (v) SEQ ID NOs: 351 and 351; SEQ ID NOs: 354 and 355; SEQ ID        NOs: 368 and 369; SEQ ID NOs: 372 and 373; SEQ ID NOs: 376 and        377; SEQ ID NOs: 386 and 387; SEQ ID NOs: 390 and 391; SEQ ID        NOs: 396 and 397; and SEQ ID NOs: 398 and 399;    -   (vi) SEQ ID NOs: 352 and 353; SEQ ID NOs: 354 and 355; SEQ ID        NOs: 370 and 371; SEQ ID NOs: 386 and 387; SEQ ID NOs: 388 and        389;    -   (vii) SEQ ID NOs: 350 and 351; SEQ ID NOs: 382 and 383; SEQ ID        NOs: 388 and 389; and SEQ ID NOs: 392 and 393;    -   (viii) SEQ ID NOs: 350 and 351; SEQ ID NOs: 366 and 367; SEQ ID        NOs: 374 and 375; SEQ ID NOs: 378 and 379; SEQ ID NOs: 382 and        383; and SEQ ID NOs: 384 and 385;    -   (ix) SEQ ID NOs: 352 and 353; SEQ ID NOs: 370 and 371; SEQ ID        NOs: 380 and 381; SEQ ID NOs: 382 and 383; and SEQ ID NOs: 386        and 387;    -   (x) SEQ ID NOs: 352 and 353; SEQ ID NOs: 356 and 357; and SEQ ID        NOs: 388 and 389;    -   (xi) SEQ ID NOs: 350 and 351; SEQ ID NOs: 354 and 355; SEQ ID        NOs: 376 and 377; and SEQ ID NOs: 386 and 387;    -   (xii) SEQ ID NOs: 350 and 351;    -   (xiii) SEQ ID NOs: 352 and 353;    -   (xiv) SEQ ID NOs: 350 and 351 and SEQ ID NOs: 378 and 379; and    -   (xv) SEQ ID NOs: 352 and 353 and SEQ ID NOs: 382 and 383, and        producing a plant from said maize germplasm, thereby producing a        drought tolerant maize plant.

In some embodiments of the presently disclosed methods, the maize plantor germplasm is of a non-naturally occurring variety of maize. In someembodiments, the genome of said maize plant or germplasm is at least 95%identical to that of NP2391.

The presently disclosed subject matter also provides in some embodimentsmethods for selecting a drought tolerant maize plant or germplasm. Insome embodiments, the methods comprise crossing a first maize plant orgermplasm with a second maize plant or germplasm, wherein said firstmaize plant or germplasm comprises within its genome a haplotypeassociated with enhanced drought tolerance, wherein said haplotype isselected from the group consisting of:

-   -   a G nucleotide at the position that corresponds to position 100        of SEQ ID NO: 2, an ACT trinucleotide at the position that        corresponds to positions 4979-4981 of SEQ ID NO: 7, a G        nucleotide at the position that corresponds to position 116 of        SEQ ID NO: 23, an A nucleotide at the position that corresponds        to position 391 of SEQ ID NO: 33, an A nucleotide at the        position that corresponds to position 472 of SEQ ID NO: 48, an A        nucleotide at the position that corresponds to position 237 of        SEQ ID NO: 56, a T nucleotide at the position that corresponds        to position 173 of SEQ ID NO: 57, and a G nucleotide at the        position that corresponds to position 267 of SEQ ID NO: 60;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, an A nucleotide at the        position that corresponds to position 309 of SEQ ID NO: 19, a G        nucleotide at the position that corresponds to position 562 of        SEQ ID NO: 25, a C nucleotide at the position that corresponds        to position 1271 of SEQ ID NO: 26, an A nucleotide at the        position that corresponds to position 266 of SEQ ID NO: 44, a C        nucleotide at the position that corresponds to position 386 of        SEQ ID NO: 46, an A nucleotide at the position that corresponds        to position 472 of SEQ ID NO: 48, and a G nucleotide at the        position that corresponds to position 111 of SEQ ID NO: 51;    -   a G nucleotide at the position that corresponds to position 100,        an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, an A nucleotide at the position that        corresponds to position 217 of SEQ ID NO: 23, a C nucleotide at        the position that corresponds to position 746 of SEQ ID NO: 24,        a C nucleotide at the position that corresponds to position 258        of SEQ ID NO: 29, an A nucleotide at the position that        corresponds to position 266 of SEQ ID NO: 44, a G nucleotide at        the position that corresponds to position 472 of SEQ ID NO: 48,        a G nucleotide at the position that corresponds to position 193        of SEQ ID NO: 55, and a C nucleotide at the position that        corresponds to position 486 of SEQ ID NO: 58;    -   a deletion at nucleotide at the position that corresponds to        positions 264-271 of SEQ ID NO: 2, an A nucleotide at the        position that corresponds to position 4641 of SEQ ID NO: 7, an A        nucleotide at the position that corresponds to position 309 of        SEQ ID NO: 19, an A nucleotide at the position that corresponds        to position 391 of SEQ ID NO: 33, a G nucleotide at the position        that corresponds to position 237 of SEQ ID NO: 56, and a C        nucleotide at the position that corresponds to position 486 of        SEQ ID NO: 58;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, a G nucleotide at the        position that corresponds to position 463 of SEQ ID NO: 19, a C        nucleotide at the position that corresponds to position 254 of        SEQ ID NO: 27, an A nucleotide at the position that corresponds        to position 391 of SEQ ID NO: 33, a T nucleotide at the position        that corresponds to position 475 of SEQ ID NO: 45, a G        nucleotide at the position that corresponds to position 193 of        SEQ ID NO: 55, a C nucleotide at the position that corresponds        to position 516 of SEQ ID NO: 56, a G nucleotide at the position        that corresponds to position 729 of SEQ ID NO: 59, and a G        nucleotide at the position that corresponds to position 267 of        SEQ ID NO: 60;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, a G nucleotide at the position that        corresponds to position 463 of SEQ ID NO: 19, a C nucleotide at        the position that corresponds to position 258 of SEQ ID NO: 29,        a G nucleotide at the position that corresponds to position 193        of SEQ ID NO: 55, and a G nucleotide at the position that        corresponds to position 237 of SEQ ID NO: 56;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, an A nucleotide at the        position that corresponds to position 472 of SEQ ID NO: 48, an A        nucleotide at the position that corresponds to position 237 of        SEQ ID NO: 56, and a T nucleotide at the position that        corresponds to position 173 of SEQ ID NO: 57;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, a C nucleotide at the        position that corresponds to position 1271 of SEQ ID NO: 26, an        A nucleotide at the position that corresponds to position 266 of        SEQ ID NO: 44, a C nucleotide at the position that corresponds        to position 386 of SEQ ID NO: 46, an A nucleotide at the        position that corresponds to position 472 of SEQ ID NO: 48, and        a G nucleotide at the position that corresponds to position 111        of SEQ ID NO: 51;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, a C nucleotide at the position that        corresponds to position 258 of SEQ ID NO: 29, a G nucleotide at        the position that corresponds to position 87 of SEQ ID NO: 47, a        G nucleotide at the position that corresponds to position 472 of        SEQ ID NO: 48, and a G nucleotide at the position that        corresponds to position 193 of SEQ ID NO: 55;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, an A nucleotide at the position that        corresponds to position 309 of SEQ ID NO: 19, and a G nucleotide        at the position that corresponds to position 237 of SEQ ID NO:        56;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, a G nucleotide at the        position that corresponds to position 463 of SEQ ID NO: 19, a T        nucleotide at the position that corresponds to position 475 of        SEQ ID NO: 45, and a G nucleotide at the position that        corresponds to position 193 of SEQ ID NO: 55;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7 and a C nucleotide at the        position that corresponds to position 386 of SEQ ID NO: 46; and    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7 and a G nucleotide at the position that        corresponds to position 472 of SEQ ID NO: 48,    -   and combinations thereof; and        selecting a progeny plant or germplasm that possesses said        haplotype within its genome, thereby selecting a drought        tolerant maize plant or germplasm. In some embodiments, either        the first maize plant or germplasm or the second maize plant or        germplasm, or both, is of a non-naturally occurring variety of        maize. In some embodiments, the genome of said first maize plant        or germplasm is at least 95% identical to that of CML333,        CML322, Cateto SP VII, Confite Morocho AYA 38, or Tuxpeno        VEN 692. In some embodiments, the first maize plant or germplasm        is selected from the group consisting of CML333, CML322, Cateto        SP VII, Confite Morocho AYA 38, and Tuxpeno VEN 692.

In some embodiments of the presently disclosed methods, the genome ofsaid second maize plant or germplasm is at least 95% identical to thatof an elite variety of maize. In some embodiments, the second maizeplant or germplasm is of an elite variety of maize. In some embodiments,the elite variety of maize is NP2391.

IV. PRODUCTION OF IMPROVED TRAIT CARRYING MAIZE PLANTS BY TRANSGENICMETHODS

In some embodiments, the presently disclosed subject matter relates tothe use of polymorphisms (including but not limited to SNPs) ortrait-conferring parts for producing a trait carrying maize plant byintroducing a nucleic acid sequence comprising a trait-associated alleleand/or haplotype of the polymorphism into a recipient plant.

A donor plant, with the nucleic acid sequence that comprises a wateroptimization trait allele and/or haplotype can be transferred to therecipient plant lacking the allele and/or the haplotype. The nucleicacid sequence can be transferred by crossing a water optimization traitcarrying donor plant with a non-trait carrying recipient plant (e.g., byintrogression), by transformation, by protoplast transformation orfusion, by a doubled haploid technique, by embryo rescue, or by anyother nucleic acid transfer system. Then, if desired, progeny plantscomprising one or more of the presently disclosed water optimizationtrait alleles and/or haplotypes can be selected. A nucleic acid sequencecomprising an water optimization trait allele and/or haplotype can beisolated from the donor plant using methods known in the art, and theisolated nucleic acid sequence can transform the recipient plant bytransgenic methods. This can occur with a vector, in a gamete, or othersuitable transfer element, such as a ballistic particle coated with thenucleic acid sequence.

Plant transformation generally involves the construction of anexpression vector that will function in plant cells and includes nucleicacid sequence that comprises an allele and/or haplotype associated withthe water optimization trait, which vector can comprise a wateroptimization trait-conferring gene. This gene usually is controlled oroperatively linked to one or more regulatory element, such as apromoter. The expression vector can contain one or more such operablylinked gene/regulatory element combinations, provided that at least oneof the genes contained in the combinations encodes water optimizationtrait. The vector(s) can be in the form of a plasmid, and can be used,alone or in combination with other plasmids, to provide transgenicplants that are better water optimization plants, using transformationmethods known in the art, such as the Agrobacterium transformationsystem.

Transformed cells often contain a selectable marker to allowtransformation identification. The selectable marker is typicallyadapted to be recovered by negative selection (by inhibiting the growthof cells that do not contain the selectable marker gene), or by positiveselection (by screening for the product encoded by the selectable markergene). Many commonly used selectable marker genes for planttransformation are known in the art, and include, for example, genesthat code for enzymes that metabolically detoxify a selective chemicalagent that can be an antibiotic or a herbicide, or genes that encode analtered target which is insensitive to the inhibitor. Several positiveselection methods are known in the art, such as mannose selection.Alternatively, marker-less transformation can be used to obtain plantswithout the aforementioned marker genes, the techniques for which arealso known in the art.

V. DROUGHT TOLERANT MAIZE PLANTS AND GERMPLASMS

The presently disclosed subject matter provides drought tolerant maizeplants and germplasms. As discussed above, the methods of the presentlydisclosed subject matter can be utilized to identify, produce and/orselect a drought tolerant maize plant or germplasm. In addition to themethods described above, a drought tolerant maize plant or germplasm canbe produced by any method whereby a marker associated with enhanceddrought tolerance is introduced into the maize plant or germplasm,including, but not limited to, transformation, protoplast transformationor fusion, a double haploid technique, embryo rescue, or by any othernucleic acid transfer system.

In some embodiments, the maize plant or germplasm comprises anon-naturally occurring variety of maize. In some embodiments, the maizeplant or germplasm is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 99% or 100% identical to that of an elite variety ofmaize. In some embodiments, the genome of said maize plant or germplasmis at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,99% or 100%, identical to that of NP2391.

The maize plant or germplasm can be the progeny of a cross between anelite variety of maize and a variety of maize that comprises an alleleassociated with enhanced drought tolerance. In some embodiments, theelite variety of maize is NP2391. In some embodiments, the genome of theelite variety of maize is at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 99% or 100% identical to that of NP2391. Insome embodiments, the variety comprising an allele associated withenhanced drought tolerance is CML333, CML322, Cateto SP VII, ConfiteMorocho AYA 38, or Tuxpeno VEN 692. In some embodiments, the genome ofthe variety comprising an allele associated with enhanced droughttolerance is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 99% or 100% identical to that of CML333, CML322, Cateto SPVII, Confite Morocho AYA 38, or Tuxpeno VEN 692.

The maize plant or germplasm can be the progeny of an introgressionwherein the recurrent parent is an elite variety of maize and the donorcomprises an allele associated with enhanced drought tolerance. In someembodiments, the recurrent parent is NP2391. In some embodiments, thegenome of the recurrent parent is at least about 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identical to that ofNP2391. In some embodiments, the donor is CML333, CML322, Cateto SP VII,Confite Morocho AYA 38, or Tuxpeno VEN 692. In some embodiments, thegenome of the donor is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 99% or 100% identical to that of CML333, CML322,Cateto SP VII, Confite Morocho AYA 38, or Tuxpeno VEN 692.

The maize plant or germplasm can be the progeny of a cross between afirst elite variety of maize (e.g., a tester line) and the progeny of across between a second elite variety of maize (e.g., a recurrent parent)and a variety of maize that comprises an allele associated with enhanceddrought tolerance (e.g., a donor). In some embodiments, the first elitevariety of maize is NP2460. In some embodiments, the genome of the firstelite variety of maize is at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 99% or 100% identical to that of NP2460. Insome embodiments, the second elite variety of maize is NP2391. In someembodiments, the genome of the second elite variety of maize is at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%identical to that of NP2391. In some embodiments, the variety comprisingan allele associated with enhanced drought tolerance is CML333, CML322,Cateto SP VII, Confite Morocho AYA 38, or Tuxpeno VEN 692. In someembodiments, the genome of the variety comprising an allele associatedwith enhanced drought tolerance is at least about 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identical to that ofCML333, CML322, Cateto SP VII, Confite Morocho AYA 38, or Tuxpeno VEN692.

The maize plant or germplasm can be the progeny of a cross between afirst elite variety of maize and the progeny of an introgression whereinthe recurrent parent is a second elite variety of maize and the donorcomprises an allele associated with enhanced drought tolerance. In someembodiments, the first elite variety of maize is NP2460. In someembodiments, the genome of the first elite variety of maize is at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%identical to that of NP2460. In some embodiments, the recurrent parentis NP2391. In some embodiments, the genome of the recurrent parent is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%or 100% identical to that of NP2391. In some embodiments, the donor isCML333, CML322, Cateto SP VII, Confite Morocho AYA 38, or Tuxpeno VEN692. In some embodiments, the genome of the donor is at least about 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identicalto that of CML333, CML322, Cateto SP VII, Confite Morocho AYA 38, orTuxpeno VEN 692.

Thus, the presently disclosed subject matter provides in someembodiments inbred Zea mays plants comprising one or more allelesassociated with a desired water optimization trait. In some embodiments:

(i) the water optimization trait is grain yield at standard moisturepercentage (YGSMN), and the favorable allele comprises a nucleotidesequence comprising an A at nucleotide position 270 of SEQ ID NO: 1; a Gat nucleotide position 216 of SEQ ID NO: 3; an A at nucleotide position503 of SEQ ID NO: 4; a CGCG tetranucleotide at nucleotide positions818-821 of SEQ ID NO: 5; a G at nucleotide position 254 of SEQ ID NO: 6;a GA dinucleotide at nucleotide positions 4497-4498 of SEQ ID NO: 7; anA at nucleotide position 4641 of SEQ ID NO: 7; a C or a T at nucleotideposition 4792 of SEQ ID NO: 7; a T at nucleotide position 4836 of SEQ IDNO: 7; an ACT or a TCC trinucleotide at nucleotide positions 4979-4981of SEQ ID NO: 7; a C at nucleotide position 292 of SEQ ID NO: 9; an A atnucleotide position 166 of SEQ ID NO: 10; a C at nucleotide position 94of SEQ ID NO: 12; a C at nucleotide position 86 of SEQ ID NO: 13; a G atnucleotide position 89 of SEQ ID NO: 13; an A at nucleotide position 753of SEQ ID NO: 15; a G at nucleotide position 755 of SEQ ID NO: 16; a Gat nucleotide position 431 of SEQ ID NO: 17; an A at nucleotide position309 of SEQ ID NO: 19; a CTGG tetranucleotide at nucleotide positions773-776 of SEQ ID NO: 20; a deletion of nucleotide positions 316-324 ofSEQ ID NO: 21; a G at nucleotide position 562 of SEQ ID NO: 25; a T atnucleotide position 254 of SEQ ID NO: 27; a T at nucleotide position 496of SEQ ID NO: 28; a G at nucleotide position 398 of SEQ ID NO: 30; a Gat nucleotide position 239 of SEQ ID NO: 31; a G at nucleotide position208 of SEQ ID NO: 32; a CA dinucleotide at the position that correspondsto positions 144-145 of SEQ ID NO: 34; a T nucleotide at the positionthat corresponds to position 169 of SEQ ID NO: 34; a G nucleotide at theposition that corresponds to position 76 of SEQ ID NO: 35; a Tnucleotide at the position that corresponds to position 698 of SEQ IDNO: 36; an A or a G nucleotide at the position that corresponds toposition 386 of SEQ ID NO: 37; a G nucleotide at the position thatcorresponds to position 445 of SEQ ID NO: 39; an A nucleotide at theposition that corresponds to position 602 of SEQ ID NO: 40; a Gnucleotide at the position that corresponds to position 190 of SEQ IDNO: 41; a C nucleotide at the position that corresponds to position 580of SEQ ID NO: 41; a TTG trinucleotide at the position that correspondsto positions 266-268 of SEQ ID NO: 42; an A nucleotide at the positionthat corresponds to position 708 of SEQ ID NO: 43; a G nucleotide at theposition that corresponds to position 650 of SEQ ID NO: 49; an A or a Tnucleotide at the position that corresponds to position 541 of SEQ IDNO: 50; a T nucleotide at the position that corresponds to position 428of SEQ ID NO: 53; a C nucleotide at the position that corresponds toposition 491 of SEQ ID NO: 53; and/or an A nucleotide at the positionthat corresponds to position 126 of SEQ ID NO: 54; and/or

(ii) the water optimization trait is grain moisture at harvest (GMSTP),and the favorable allele comprises a nucleotide sequence comprising an Anucleotide at the position that corresponds to position 254 of SEQ IDNO: 6; an A nucleotide at the position that corresponds to position 217of SEQ ID NO: 8; a C nucleotide at the position that corresponds toposition 292 of SEQ ID NO: 9; an A nucleotide at the position thatcorresponds to position 166 of SEQ ID NO: 10; a G nucleotide at theposition that corresponds to position 148 of SEQ ID NO: 11; an Anucleotide at the position that corresponds to position 35 of SEQ ID NO:13; a G nucleotide at the position that corresponds to position 432 ofSEQ ID NO: 14; a G nucleotide at the position that corresponds toposition 518 of SEQ ID NO: 18; an A nucleotide at the position thatcorresponds to position 182 of SEQ ID NO: 19; a C nucleotide at theposition that corresponds to position 463 of SEQ ID NO: 19; a CTGGtetranucleotide at the position that corresponds to positions 773-776 ofSEQ ID NO: 20; a G nucleotide at the position that corresponds toposition 211 of SEQ ID NO: 22; a G nucleotide at the position thatcorresponds to position 562 of SEQ ID NO: 25; a C nucleotide at theposition that corresponds to position 254 of SEQ ID NO: 27; a Gnucleotide at the position that corresponds to position 239 of SEQ IDNO: 31; a CA dinucleotide at the position that corresponds to positions144-145 of SEQ ID NO: 34; an A nucleotide at the position thatcorresponds to position 537 of SEQ ID NO: 34; an A nucleotide at theposition that corresponds to position 386 of SEQ ID NO: 37; a Cnucleotide at the position that corresponds to position 309 of SEQ IDNO: 38; an A nucleotide at the position that corresponds to position 342of SEQ ID NO: 38; a C nucleotide at the position that corresponds toposition 445 of SEQ ID NO: 39; an A nucleotide at the position thatcorresponds to position 190 of SEQ ID NO: 41; a C nucleotide at theposition that corresponds to position 708 of SEQ ID NO: 43; a Gnucleotide at the position that corresponds to position 650 of SEQ IDNO: 49; a C nucleotide at the position that corresponds to position 428of SEQ ID NO: 53; or a C nucleotide at the position that corresponds toposition 491 of SEQ ID NO: 53; and/or

(iii) the water optimization trait is grain weight per plot (GWTPN), andthe favorable allele comprises a nucleotide sequence comprising a T atnucleotide position 518 of SEQ ID NO: 18.

The presently disclosed subject matter also provides in some embodimentsZea mays plants comprising at least one favorable allele contributing towater optimization, which allele is defined by at least one markerallele comprising a polymorphic site and characterized by a PCRamplification product obtainable in a PCR reaction using a PCRoligonucleotide primer or a plurality of oligonucleotide primers,particularly a pair of PCR oligonucleotide primers or a plurality ofprimer pairs, but especially a primer pair selected from the groupconsisting of primer pair 1 represented by a primer comprising SEQ IDNO: 118 and a primer comprising SEQ ID NO: 119; primer pair 2represented by a primer comprising SEQ ID NO: 120 and a primercomprising SEQ ID NO: 121; primer pair 3 represented by a primercomprising SEQ ID NO: 122 and a primer comprising SEQ ID NO: 123; primerpair 4 represented by a primer comprising SEQ ID NO: 124 and a primercomprising SEQ ID NO: 125; primer pair 5 represented by a primercomprising SEQ ID NO: 126 and a primer comprising SEQ ID NO: 127; primerpair 6 represented by a primer comprising SEQ ID NO: 128 and a primercomprising SEQ ID NO: 129; primer pair 7 represented by a primercomprising SEQ ID NO: 130 and a primer comprising SEQ ID NO: 131; primerpair 8 represented by a primer comprising SEQ ID NO: 132 and a primercomprising SEQ ID NO: 133; primer pair 9 represented by a primercomprising SEQ ID NO: 134 and a primer comprising SEQ ID NO: 135; primerpair 10 represented by a primer comprising SEQ ID NO: 136 and a primercomprising SEQ ID NO: 137; primer pair 11 represented by a primercomprising SEQ ID NO: 138 and a primer comprising SEQ ID NO: 139; primerpair 12 represented by a primer comprising SEQ ID NO: 140 and a primercomprising SEQ ID NO: 141; primer pair 13 represented by a primercomprising SEQ ID NO: 142 and a primer comprising SEQ ID NO: 143; primerpair 14 represented by a primer comprising SEQ ID NO: 144 and a primercomprising SEQ ID NO: 145; primer pair 15 represented by a primercomprising SEQ ID NO: 146 and a primer comprising SEQ ID NO: 147; primerpair 16 represented by a primer comprising SEQ ID NO: 148 and a primercomprising SEQ ID NO: 149; primer pair 17 represented by a primercomprising SEQ ID NO: 150 and a primer comprising SEQ ID NO: 151; primerpair 18 represented by a primer comprising SEQ ID NO: 152 and a primercomprising SEQ ID NO: 153; primer pair 19 represented by a primercomprising SEQ ID NO: 154 and a primer comprising SEQ ID NO: 155; primerpair 20 represented by a primer comprising SEQ ID NO: 156 and a primercomprising SEQ ID NO: 157; primer pair 21 represented by a primercomprising SEQ ID NO: 158 and a primer comprising SEQ ID NO: 159; primerpair 22 represented by a primer comprising SEQ ID NO: 160 and a primercomprising SEQ ID NO: 161; primer pair 23 represented by a primercomprising SEQ ID NO: 162 and a primer comprising SEQ ID NO: 163; primerpair 24 represented by a primer comprising SEQ ID NO: 164 and a primercomprising SEQ ID NO: 165; primer pair 25 represented by a primercomprising SEQ ID NO: 166 and a primer comprising SEQ ID NO: 167; primerpair 26 represented by a primer comprising SEQ ID NO: 168 and a primercomprising SEQ ID NO: 169; primer pair 27 represented by a primercomprising SEQ ID NO: 170 and a primer comprising SEQ ID NO: 171; primerpair 28 represented by a primer comprising SEQ ID NO: 172 and a primercomprising SEQ ID NO: 173; primer pair 29 represented by a primercomprising SEQ ID NO: 174 and a primer comprising SEQ ID NO: 175; primerpair 30 represented by a primer comprising SEQ ID NO: 176 and a primercomprising SEQ ID NO: 177; primer pair 31 represented by a primercomprising SEQ ID NO: 178 and a primer comprising SEQ ID NO: 179; primerpair 32 represented by a primer comprising SEQ ID NO: 180 and a primercomprising SEQ ID NO: 181; primer pair 33 represented by a primercomprising SEQ ID NO: 182 and a primer comprising SEQ ID NO: 183; primerpair 34 represented by a primer comprising SEQ ID NO: 184 and a primercomprising SEQ ID NO: 185; primer pair 35 represented by a primercomprising SEQ ID NO: 186 and a primer comprising SEQ ID NO: 187; primerpair 36 represented by a primer comprising SEQ ID NO: 188 and a primercomprising SEQ ID NO: 189; primer pair 37 represented by a primercomprising SEQ ID NO: 190 and a primer comprising SEQ ID NO: 191; primerpair 38 represented by a primer comprising SEQ ID NO: 192 and a primercomprising SEQ ID NO: 193; primer pair 39 represented by a primercomprising SEQ ID NO: 194 and a primer comprising SEQ ID NO: 195; primerpair 40 represented by a primer comprising SEQ ID NO: 196 and a primercomprising SEQ ID NO: 197; primer pair 41 represented by a primercomprising SEQ ID NO: 198 and a primer comprising SEQ ID NO: 199; primerpair 42 represented by a primer comprising SEQ ID NO: 200 and a primercomprising SEQ ID NO: 201; primer pair 43 represented by a primercomprising SEQ ID NO: 202 and a primer comprising SEQ ID NO: 203; primerpair 44 represented by a primer comprising SEQ ID NO: 204 and a primercomprising SEQ ID NO: 205; primer pair 45 represented by a primercomprising SEQ ID NO: 206 and a primer comprising SEQ ID NO: 207; primerpair 46 represented by a primer comprising SEQ ID NO: 208 and a primercomprising SEQ ID NO: 209; primer pair 47 represented by a primercomprising SEQ ID NO: 210 and a primer comprising SEQ ID NO: 211; primerpair 48 represented by a primer comprising SEQ ID NO: 212 and a primercomprising SEQ ID NO: 213; primer pair 49 represented by a primercomprising SEQ ID NO: 214 and a primer comprising SEQ ID NO: 215; primerpair 50 represented by a primer comprising SEQ ID NO: 216 and a primercomprising SEQ ID NO: 217; primer pair 51 represented by a primercomprising SEQ ID NO: 218 and a primer comprising SEQ ID NO: 219; primerpair 52 represented by a primer comprising SEQ ID NO: 220 and a primercomprising SEQ ID NO: 221; primer pair 53 represented by a primercomprising SEQ ID NO: 222 and a primer comprising SEQ ID NO: 223; primerpair 54 represented by a primer comprising SEQ ID NO: 224 and a primercomprising SEQ ID NO: 225; primer pair 55 represented by a primercomprising SEQ ID NO: 226 and a primer comprising SEQ ID NO: 227; primerpair 56 represented by a primer comprising SEQ ID NO: 228 and a primercomprising SEQ ID NO: 229; and primer pair 57 represented by a primercomprising SEQ ID NO: 230 and a primer comprising SEQ ID NO: 231.

In some embodiments, the presently disclosed subject matter provides anon-naturally occurring maize plant or germplasm having in its genome ahaplotype associated with enhanced drought tolerance. In someembodiments, wherein said haplotype is selected from the groupconsisting of:

-   -   a G nucleotide at the position that corresponds to position 100        of SEQ ID NO: 2, an ACT trinucleotide at the position that        corresponds to positions 4979-4981 of SEQ ID NO: 7, a G        nucleotide at the position that corresponds to position 116 of        SEQ ID NO: 23, an A nucleotide at the position that corresponds        to position 391 of SEQ ID NO: 33, an A nucleotide at the        position that corresponds to position 472 of SEQ ID NO: 48, an A        nucleotide at the position that corresponds to position 237 of        SEQ ID NO: 56, a T nucleotide at the position that corresponds        to position 173 of SEQ ID NO: 57, and a G nucleotide at the        position that corresponds to position 267 of SEQ ID NO: 60;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, an A nucleotide at the        position that corresponds to position 309 of SEQ ID NO: 19, a G        nucleotide at the position that corresponds to position 562 of        SEQ ID NO: 25, a C nucleotide at the position that corresponds        to position 1271 of SEQ ID NO: 26, an A nucleotide at the        position that corresponds to position 266 of SEQ ID NO: 44, a C        nucleotide at the position that corresponds to position 386 of        SEQ ID NO: 46, an A nucleotide at the position that corresponds        to position 472 of SEQ ID NO: 48, and a G nucleotide at the        position that corresponds to position 111 of SEQ ID NO: 51;    -   a G nucleotide at the position that corresponds to position 100,        an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, an A nucleotide at the position that        corresponds to position 217 of SEQ ID NO: 23, a C nucleotide at        the position that corresponds to position 746 of SEQ ID NO: 24,        a C nucleotide at the position that corresponds to position 258        of SEQ ID NO: 29, an A nucleotide at the position that        corresponds to position 266 of SEQ ID NO: 44, a G nucleotide at        the position that corresponds to position 472 of SEQ ID NO: 48,        a G nucleotide at the position that corresponds to position 193        of SEQ ID NO: 55, and a C nucleotide at the position that        corresponds to position 486 of SEQ ID NO: 58;    -   a deletion at nucleotide at the position that corresponds to        positions 264-271 of SEQ ID NO: 2, an A nucleotide at the        position that corresponds to position 4641 of SEQ ID NO: 7, an A        nucleotide at the position that corresponds to position 309 of        SEQ ID NO: 19, an A nucleotide at the position that corresponds        to position 391 of SEQ ID NO: 33, a G nucleotide at the position        that corresponds to position 237 of SEQ ID NO: 56, and a C        nucleotide at the position that corresponds to position 486 of        SEQ ID NO: 58;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, a G nucleotide at the        position that corresponds to position 463 of SEQ ID NO: 19, a C        nucleotide at the position that corresponds to position 254 of        SEQ ID NO: 27, an A nucleotide at the position that corresponds        to position 391 of SEQ ID NO: 33, a T nucleotide at the position        that corresponds to position 475 of SEQ ID NO: 45, a G        nucleotide at the position that corresponds to position 193 of        SEQ ID NO: 55, a C nucleotide at the position that corresponds        to position 516 of SEQ ID NO: 56, a G nucleotide at the position        that corresponds to position 729 of SEQ ID NO: 59, and a G        nucleotide at the position that corresponds to position 267 of        SEQ ID NO: 60;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, a G nucleotide at the position that        corresponds to position 463 of SEQ ID NO: 19, a C nucleotide at        the position that corresponds to position 258 of SEQ ID NO: 29,        a G nucleotide at the position that corresponds to position 193        of SEQ ID NO: 55, and a G nucleotide at the position that        corresponds to position 237 of SEQ ID NO: 56;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, an A nucleotide at the        position that corresponds to position 472 of SEQ ID NO: 48, an A        nucleotide at the position that corresponds to position 237 of        SEQ ID NO: 56, and a T nucleotide at the position that        corresponds to position 173 of SEQ ID NO: 57;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, a C nucleotide at the        position that corresponds to position 1271 of SEQ ID NO: 26, an        A nucleotide at the position that corresponds to position 266 of        SEQ ID NO: 44, a C nucleotide at the position that corresponds        to position 386 of SEQ ID NO: 46, an A nucleotide at the        position that corresponds to position 472 of SEQ ID NO: 48, and        a G nucleotide at the position that corresponds to position 111        of SEQ ID NO: 51;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, a C nucleotide at the position that        corresponds to position 258 of SEQ ID NO: 29, a G nucleotide at        the position that corresponds to position 87 of SEQ ID NO: 47, a        G nucleotide at the position that corresponds to position 472 of        SEQ ID NO: 48, and a G nucleotide at the position that        corresponds to position 193 of SEQ ID NO: 55;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7, an A nucleotide at the position that        corresponds to position 309 of SEQ ID NO: 19, and a G nucleotide        at the position that corresponds to position 237 of SEQ ID NO:        56;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7, a G nucleotide at the        position that corresponds to position 463 of SEQ ID NO: 19, a T        nucleotide at the position that corresponds to position 475 of        SEQ ID NO: 45, and a G nucleotide at the position that        corresponds to position 193 of SEQ ID NO: 55;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7;    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7;    -   an ACT trinucleotide at the position that corresponds to        positions 4979-4981 of SEQ ID NO: 7 and a C nucleotide at the        position that corresponds to position 386 of SEQ ID NO: 46; and    -   an A nucleotide at the position that corresponds to position        4641 of SEQ ID NO: 7 and a G nucleotide at the position that        corresponds to position 472 of SEQ ID NO: 48,    -   and combinations thereof.

In some embodiments, the genome of said maize plant or germplasm is atleast 95% identical to that of an elite variety of maize. In someembodiments, the elite variety of maize is NP2391. In some embodiments,the maize plant or germplasm is derived from crossing an elite varietyof maize with an exotic variety of maize. In some embodiments, thegenome of said elite variety of maize is at least 95% identical to thatof NP2391. In some embodiments, the elite variety of maize is NP2391.

In some embodiments, the genome of said exotic variety of maize is atleast 95% identical to that of CML333, CML322, Cateto SP VII, ConfiteMorocho AYA 38, or Tuxpeno VEN 692. In some embodiments, 32. In someembodiments, said maize plant or germplasm is derived from crossing afirst elite variety of maize with the progeny of a cross between asecond elite variety of maize and an exotic variety of maize.

In some embodiments, the genome of said first elite variety of maize isat least 95% identical to that of NP2460. In some embodiments, the firstelite variety of maize is NP2460.

In some embodiments, the genome of said second elite variety of maize isat least 95% identical to that of NP2391. In some embodiments, thesecond elite variety of maize is NP2391.

In some embodiments, the genome of said exotic variety of maize is atleast 95% identical to CML333, CML322, Cateto SP VII, Confite MorochoAYA 38, or Tuxpeno VEN 692. In some embodiments, said exotic variety ofmaize is selected from the group consisting of CML333, CML322, Cateto SPVII, Confite Morocho AYA 38, and Tuxpeno VEN 692.

The presently disclosed subject matter also provides in some embodimentsgrain and/or kernels produced by from a maize plant described herein.

VI. OTHER COMPOSITIONS

In some embodiments, the presently disclosed subject matter alsoprovides pairs of primers consisting of a forward primer and a reverseprimer which primers are capable of amplifying in a PCR reaction afragment of the marker allele, which is genetically linked to oridentical with the favorable allele contributing to a water optimizationphenotype, wherein said marker allele comprises a polymorphism, whichpolymorphism is diagnostic for the favorable allele. In someembodiments, the primer pairs are selected from the group consisting ofprimer pair 1 represented by a primer comprising SEQ ID NO: 118 and aprimer comprising SEQ ID NO: 119; primer pair 2 represented by a primercomprising SEQ ID NO: 120 and a primer comprising SEQ ID NO: 121; primerpair 3 represented by a primer comprising SEQ ID NO: 122 and a primercomprising SEQ ID NO: 123; primer pair 4 represented by a primercomprising SEQ ID NO: 124 and a primer comprising SEQ ID NO: 125; primerpair 5 represented by a primer comprising SEQ ID NO: 126 and a primercomprising SEQ ID NO: 127; primer pair 6 represented by a primercomprising SEQ ID NO: 128 and a primer comprising SEQ ID NO: 129; primerpair 7 represented by a primer comprising SEQ ID NO: 130 and a primercomprising SEQ ID NO: 131; primer pair 8 represented by a primercomprising SEQ ID NO: 132 and a primer comprising SEQ ID NO: 133; primerpair 9 represented by a primer comprising SEQ ID NO: 134 and a primercomprising SEQ ID NO: 135; primer pair 10 represented by a primercomprising SEQ ID NO: 136 and a primer comprising SEQ ID NO: 137; primerpair 11 represented by a primer comprising SEQ ID NO: 138 and a primercomprising SEQ ID NO: 139; primer pair 12 represented by a primercomprising SEQ ID NO: 140 and a primer comprising SEQ ID NO: 141; primerpair 13 represented by a primer comprising SEQ ID NO: 142 and a primercomprising SEQ ID NO: 143; primer pair 14 represented by a primercomprising SEQ ID NO: 144 and a primer comprising SEQ ID NO: 145; primerpair 15 represented by a primer comprising SEQ ID NO: 146 and a primercomprising SEQ ID NO: 147; primer pair 16 represented by a primercomprising SEQ ID NO: 148 and a primer comprising SEQ ID NO: 149; primerpair 17 represented by a primer comprising SEQ ID NO: 150 and a primercomprising SEQ ID NO: 151; primer pair 18 represented by a primercomprising SEQ ID NO: 152 and a primer comprising SEQ ID NO: 153; primerpair 19 represented by a primer comprising SEQ ID NO: 154 and a primercomprising SEQ ID NO: 155; primer pair 20 represented by a primercomprising SEQ ID NO: 156 and a primer comprising SEQ ID NO: 157; primerpair 21 represented by a primer comprising SEQ ID NO: 158 and a primercomprising SEQ ID NO: 159; primer pair 22 represented by a primercomprising SEQ ID NO: 160 and a primer comprising SEQ ID NO: 161; primerpair 23 represented by a primer comprising SEQ ID NO: 162 and a primercomprising SEQ ID NO: 163; primer pair 24 represented by a primercomprising SEQ ID NO: 164 and a primer comprising SEQ ID NO: 165; primerpair 25 represented by a primer comprising SEQ ID NO: 166 and a primercomprising SEQ ID NO: 167; primer pair 26 represented by a primercomprising SEQ ID NO: 168 and a primer comprising SEQ ID NO: 169; primerpair 27 represented by a primer comprising SEQ ID NO: 170 and a primercomprising SEQ ID NO: 171; primer pair 28 represented by a primercomprising SEQ ID NO: 172 and a primer comprising SEQ ID NO: 173; primerpair 29 represented by a primer comprising SEQ ID NO: 174 and a primercomprising SEQ ID NO: 175; primer pair 30 represented by a primercomprising SEQ ID NO: 176 and a primer comprising SEQ ID NO: 177; primerpair 31 represented by a primer comprising SEQ ID NO: 178 and a primercomprising SEQ ID NO: 179; primer pair 32 represented by a primercomprising SEQ ID NO: 180 and a primer comprising SEQ ID NO: 181; primerpair 33 represented by a primer comprising SEQ ID NO: 182 and a primercomprising SEQ ID NO: 183; primer pair 34 represented by a primercomprising SEQ ID NO: 184 and a primer comprising SEQ ID NO: 185; primerpair 35 represented by a primer comprising SEQ ID NO: 186 and a primercomprising SEQ ID NO: 187; primer pair 36 represented by a primercomprising SEQ ID NO: 188 and a primer comprising SEQ ID NO: 189; primerpair 37 represented by a primer comprising SEQ ID NO: 190 and a primercomprising SEQ ID NO: 191; primer pair 38 represented by a primercomprising SEQ ID NO: 192 and a primer comprising SEQ ID NO: 193; primerpair 39 represented by a primer comprising SEQ ID NO: 194 and a primercomprising SEQ ID NO: 195; primer pair 40 represented by a primercomprising SEQ ID NO: 196 and a primer comprising SEQ ID NO: 197; primerpair 41 represented by a primer comprising SEQ ID NO: 198 and a primercomprising SEQ ID NO: 199; primer pair 42 represented by a primercomprising SEQ ID NO: 200 and a primer comprising SEQ ID NO: 201; primerpair 43 represented by a primer comprising SEQ ID NO: 202 and a primercomprising SEQ ID NO: 203; primer pair 44 represented by a primercomprising SEQ ID NO: 204 and a primer comprising SEQ ID NO: 205; primerpair 45 represented by a primer comprising SEQ ID NO: 206 and a primercomprising SEQ ID NO: 207; primer pair 46 represented by a primercomprising SEQ ID NO: 208 and a primer comprising SEQ ID NO: 209; primerpair 47 represented by a primer comprising SEQ ID NO: 210 and a primercomprising SEQ ID NO: 211; primer pair 48 represented by a primercomprising SEQ ID NO: 212 and a primer comprising SEQ ID NO: 213; primerpair 49 represented by a primer comprising SEQ ID NO: 214 and a primercomprising SEQ ID NO: 215; primer pair 50 represented by a primercomprising SEQ ID NO: 216 and a primer comprising SEQ ID NO: 217; primerpair 51 represented by a primer comprising SEQ ID NO: 218 and a primercomprising SEQ ID NO: 219; primer pair 52 represented by a primercomprising SEQ ID NO: 220 and a primer comprising SEQ ID NO: 221; primerpair 53 represented by a primer comprising SEQ ID NO: 222 and a primercomprising SEQ ID NO: 223; primer pair 54 represented by a primercomprising SEQ ID NO: 224 and a primer comprising SEQ ID NO: 225; primerpair 55 represented by a primer comprising SEQ ID NO: 226 and a primercomprising SEQ ID NO: 227; primer pair 56 represented by a primercomprising SEQ ID NO: 228 and a primer comprising SEQ ID NO: 229; andprimer pair 57 represented by a primer comprising SEQ ID NO: 230 and aprimer comprising SEQ ID NO: 231.

EXAMPLES

The following Examples provide illustrative embodiments. In light of thepresent disclosure and the general level of skill in the art, those ofskill will appreciate that the following Examples are intended to beexemplary only and that numerous changes, modifications, and alterationscan be employed without departing from the scope of the presentlydisclosed subject matter.

Introduction to the Examples

To assess the value of alleles under drought stress, diverse germplasmwas screened in controlled field-experiments comprising a fullirrigation control treatment and a limited irrigation treatment. Thegoal of the full irrigation treatment is to ensure water does not limitthe productivity of the crop. In contrast, the goal of the limitedirrigation treatment is to ensure that water becomes the major limitingconstraint to grain yield. Main effects (e.g., treatment and genotype)and interactions (e.g., genotype×treatment) can be determined when thetwo treatments are applied adjacent to one another in the field.Moreover, drought related phenotypes can be quantified for each genotypein the panel thereby allowing for marker:trait associations to beconducted.

In practice, the method for the limited irrigation treatment can varywidely depending upon the germplasm being screened, the soil type,climatic conditions at the site, pre-season water supply, and in-seasonwater supply, to name just a few. Initially, a site is identified wherein-season precipitation is low (to minimize the chance of unintendedwater application) and is suitable for cropping. In addition,determining the timing of the stress can be important, such that atarget is defined to ensure that year-to-year, or location-to-location,screening consistency is in place. An understanding of the treatmentintensity, or in some cases the yield loss desired from the limitedirrigation treatment, can also be considered. Selection of a treatmentintensity that is too light can fail to reveal genotypic variation.Selection of a treatment intensity that is too heavy can create largeexperimental error. Once the timing of stress is identified andtreatment intensity is described, irrigation can be managed in a mannerthat is consistent with these targets.

General methods for assessing and assessing drought tolerance can befound in Salekdeh et al., 2009 and in U.S. Pat. Nos. 6,635,803;7,314,757; 7,332,651; and 7,432,416.

Example 1 Assessment of the Phenotypic Data

In order to identify alleles that were associated with wateroptimization, hybrids were grown in different stages at multiplelocations and evaluated for water optimization. In this analysis, fourtraits were tested in stage 2-3: YGSMN (grain yield at standard moisture%), GMSTP (grain moisture at harvest), GWTPN (grain weight per plot),and PYREC (percentage yield recovery). The distribution of thephenotypic data of hybrids of the lines across locations and testers forYGSMN, GMSTP, GWTPN, and PYREC was determined. The mean values forYGSMN, GMSTP, and GWTPN were 165.41 bushels/acre, 18.94%, and 20.0bushels/plot respectively. The phenotypic data for the selected trialsincluded information from 4 locations. The number of observations inthese locations ranged from 311 to 1456. A total of 575 inbreds wereevaluated in crosses with up to 47 different inbred testers. The numberof observations for inbred lines crossed to a particular tester rangedfrom 242 to 575 across all locations.

The testing for associations between potential markers and these threetraits employed two analytical approaches: a Mixed LinearModels—(TASSEL) and a Quantitative Inbred Pedigree Disequilibrium Test(referred to herein as “QIPDT2”).

Example 2 Phenotypic Adjustments

The use of stage 2-3 data for association mapping is not a traditionalapproach, and there are several aspects of its analysis that needed tobe considered. Moreover, hybrids with various testers, instead of thelines per se, were employed for phenotyping, while both of thestatistical approaches (TASSEL and QIPDT2) were designed for data oninbred lines which require a unique trait value for each line. To obtaina unique trait value for each inbred line that could be compared againstits genotype, it was necessary to make phenotypic adjustments that helpto control the effect of tester and/or location. Additional factors(e.g., maturity group) were not considered to avoid the furtherreduction of degrees of freedom or subsets sample sizes.

To do the phenotypic adjustments, mixed linear model analyses wereperformed in two different statistical packages, SAS/JMP and R, whichwere intended to ensure that the mixed-model approaches for the largedata set were implemented correctly. Since approaches gave very closeresults, the SAS/JMP results were used for the downstream data analysis.

The “full model” analysis included effects of both locations and testersin the model as follows:Phenotype=Location effect(random)+Line effect(random)+Testereffect(fixed)+error term

The “by Location” model was used for each of the 4 selected locations asfollows:Phenotype=Line effect(random)+Tester effect(fixed)+error term

The “by Tester” model was used for each of the 4 selected subsets oflines crossed to a particular tester as follows:Phenotype=Location effect(random)+Line effect(random)+error term

The models were evaluated for convergence, estimation of covarianceestimates, significance of fixed effects, etc. Best linear unbiasedpredictors (BLUPs) for line effects were used as adjusted genotypes. Insome cases, the proposed mixed models did not converge or there was aproblem with the estimation of line effects due to the lack ofreplications. For each such case, the effect of the line was removedfrom the model and the residuals were used as a rough method to captureline effects (additional replication was obtained later in theassociation analysis where each biallelic locus was represented by thetotal number of inbred lines of each group).

The solution for the lines random effects (BLUPs) were obtained from themixed models that converged.

Example 3 Genotypic Data

A total of 2189 lines for which phenotypic data was collected in any ofthe selected trials were also genotyped. A total of 95 polymorphismscorresponding to about 57 candidate genes were scored in the inbredlines. After eliminating monomorphic assays and SNPs with allelefrequencies less than 0.01, 85 candidate polymorphisms were tested forassociation in TASSEL. Besides, 153 random polymorphisms were genotypedin the inbred lines. After filtering, 149 random polymorphisms were alsoanalyzed for association in TASSEL as anonymous candidates.

Example 4 Methodologies for Association Analysis

Association mapping (often referred as linkage disequilibrium mapping)has become a powerful tool to unveil the genetic control of complextraits. Association mapping relies on the large number of generations,and therefore recombination opportunities, in the history of a species,that allow the removal of association between a QTL and any marker nottightly linked to it (Jannink & Walsh, 2001). One of the most importantsteps in association mapping analysis is the control for populationstructure. Population structure can cause spurious correlations betweenmarkers and phenotypes, increasing the false-positive rate.

Kinship Analysis.

The method implemented in TASSEL uses a kinship matrix in themixed-model approach for controlling genetic correlations among lines.Kinship analysis was done using genotypic data on the 153 random SNPassays. A method to estimate kinship relationships based on Zhao et al.,2007 was adopted. Scripts were created to calculate Kinship coefficientsthat were defined simply as the proportion of shared alleles for eachpair of individuals (K pShared). Zhao et al. used the proportion ofshared haplotypes as their kinship coefficients. The matrix of Kcoefficients was included for some association models in TASSEL toassess the control for spurious associations due to closeinterrelatedness of the lines in the panel.

Kinship Coefficient Matrix Calculator.

The K matrix was calculated for a set of inbred lines. The kinshipcoefficient kij was calculated as proportion of shared alleles for allloci between two lines i and j, and kij=kji, kii=1.

Population Structure Analysis.

Analysis with the software program Structure (Pritchard et al., 2000)was done using genotypic data of the 153 random SNP assays.

A linkage model that incorporated population admixture and linkagebetween the markers was employed. The likelihoods of populationstructures ranging from k=1 to 15 subpopulations were determined using aburnin period of 50,000 followed by 50,000 MCMC reps. Four replicationswere run for each value of k. The estimated log probability of dataPr(X|K) for each value of k was plotted to choose an appropriate numberof subpopulations to include in the covariance matrix.

The probability for a determinate k increased along with the number of ktested. k=10 was used as the number of subpopulations for associationanalysis. The inferred ancestry table containing the fraction of eachsubpopulation contributing to the ancestry of each inbred was used as aseries of covariates in the association testing model.

Principal Component Analysis.

Principal Component Analysis (PCA) or “Eigen analysis” was used as analternative to Structure for inferring population structure fromgenotypic data. PCA has some advantages over Structure such as theability to handle large datasets in much shorter periods of time, andavoiding the need of selecting a specific number of sub-populations. PCAwas performed using the software SMARTPCA that is part of Eigenstrat(Price et al., 2006). Ten Eigenvectors and their correspondingEigenvalues for each of the lines were used as another covariate seriesfor the association models of TASSEL.

Example 5 Association Analysis Using TASSEL

Association Models in TASSEL.

The different models employed in TASSEL are shown in the Table 6. Forthe YGSMN and GMSTP phenotypes adjusted across locations and testers,the six (6) models were run and compared. Only Model 4 was run for allthe sub-sets by location and by tester.

TABLE 6 Association Models Employed in TASSEL General Lineal Models 1)Adj. Phenotype = Marker 2) Adj. Phenotype = Marker + Q (Structure) 3)Adj. Phenotype = Marker + PCA (Eigenvalues) Mixed Lineal Models 4) Adj.Phenotype = Marker + K (pshared)* 5) Adj. Phenotype = Marker + K(pshared) + Q (Structure) 6) Adj. Phenotype = Marker + K (pshared) + PCA(Eigenvalues)

The GLM procedure in TASSEL employed an option to perform permutationsto find out the experiment-wise error rate that corrected foraccumulation of false positives when doing multiple comparisons. A totalof 10,000 permutations were used for the water optimization data. TheMLM procedure did not include correction for multiple testing. TheBonferroni correction was used a posteriori to avoid accumulation offalse positives.

Example 6 Association Analysis Using QIPDT2

QIPDT2 (Quantitative Inbred Pedigree Disequilibrium Test 2) was used forassociation mapping that takes advantage of inbred pedigree information,which can give higher statistical power and lower false positive rateswith a better control of population structure issue (Stich et al. 2006,TAG 113:1121-1130). This is an extension of QIPDT originally developedfor mapping human disease genes (Zhang et al., 2001. Genetic Epidemiol21:370-375—see reference in Stich et al. 2006). An advantage of QIPDT2is that this method can be more easily applied to materials from earlybreeding stages (e.g., stage 2 and 3) because phenotypic data on thesematerials have been collected for breeding purposes. Generally speaking,the materials from early breeding stages are similar to the lines in thewell-known nested association populations (NAM), which was designed touse both linkage and linkage disequilibrium for mapping QTL.

The original QIPDT is a test statistic, T, which is calculated in thefollowing way (Stich et al. 2006):

${T = \frac{\sum\limits_{k = 1}^{p}\; D_{k}}{\sqrt{\sum\limits_{k = 1}^{p}\; D_{k}^{2}}}},{{following}\mspace{14mu}{N( {0,1} )}\mspace{14mu}{under}\mspace{14mu} H_{0}}$${D_{k} = {\sum\limits_{i = 1}^{n_{k}}\; U_{jk}}},{{E( {\sum\limits_{k = 1}^{p}\; D_{k}} )} = {0\mspace{14mu}{under}\mspace{14mu} H_{0}}}$$U_{jk} = {\sum\limits_{i = 1}^{t_{jk}}\;{( {Y_{ijk} - \overset{\_}{Y_{k}}} )X_{ijk}}}$$\overset{\_}{Y_{k}} - {{mean}\mspace{14mu}{trait}\mspace{14mu}{value}\mspace{14mu}{for}\mspace{14mu}{extended}\mspace{14mu}{pedigree}\mspace{14mu} k}$X_(ijk) − marker  value (−1, 0, 1)In the general approach, a T value is calculated for each SNP, and its pvalue is found from standard normal distribution. While this approach isuseful for testing the statistical significance of association, it doesnot provide an estimate of the magnitude of the SNP genetic effect, northe relative genetic contribution to the total phenotypic variance.

Thus, the general QIPDT approach was improved using a regression model,which is referred to herein as “QIPDT2”; the original method is thencalled QIPDT1. The model for QIPDT2 can be written as:y _(ki)=β₀+β₁ x _(ki) +e _(ki),where y_(ki) is adjusted phenotypic value for individual i in pedigreek; x_(ki) is coded marker genotypic value; β₀ is intercept; β₁ isregression coefficient, or genetic effect, of the SNP in question. Notethat the methods for adjusting phenotypic values and coding markergenotypes are the same as used by Stich et al., 2006. With this model,both the genetic effect and R² for each SNP can be estimated. It isimportant to note that the phenotypic data were pre-adjusted forexcluding effects from testers and/or locations before being furtheradjusted for pedigree structure; this adjustment was necessary toimplement the complex model in QIPDT2. The methods for pre-adjustmentwere the same as described previously for the TASSEL analysis.

Association Models in QIPDT2.

Association results from both QIPDT1 and QIPDT2 for the whole data setand split subsets for locations and testers were generated. Like theanalysis with TASSEL, the phenotypic data were adjusted for locationsand/or testers, depending on which subset was used. This resulted in oneadjusted phenotypic value (either BLUP line values or model residuals)for each inbred, which contains a combination of all genetic effects forthe inbred and random residual only.

Before QIPDT analysis, all inbreds were grouped into different nuclearfamilies, according to their parental lines. The use of nuclear familieswas expected to give better control of population structure thanextended pedigrees that were used in Stich et al., 2006. For QIPDT1, atest statistic (Z value) and corresponding p value were estimated foreach SNP; for QIPDT2, a test statistic (T value) and corresponding pvalue were derived from the simple regression model, along with Rsquare, for each SNP. QIPDT2 was more powerful than QIPDT1, in terms ofp values. Since QIPDT2 also gave estimates (R²) for relativecontribution for each SNP, QIPDT2 was used for reporting associationresults from the QIPDT approach.

Example 7 Significance and Contributions of Favorable Alleles to WaterOptimization Phenotypes

P values and contributions that each favorable allele was observed tohave on the water optimization phenotypes YGSMN, GMSTP, and GWTPN werecalculated. These values are summarized in Tables 7-9. In Tables 7-9,the term “contribution” refers to the contribution that the favorableallele was calculated to have with respect to the phenotype observed inview of the mean values of 201.68 bushels/acre, 18.95%, and 25.29bushels/plot for YGSMN, GMSTP, and GWTPN, respectively. In Tables 7-9,the “contribution” is expressed in bushels/acre, percent, andbushels/plot for YGSMN, GMSTP, and GWTPN, respectively.

TABLE 7 Contributions of Favorable Alleles to Increased Wateroptimization Identified by Both TASSEL and QIPDT2 SNP SEQ ID NO.Position F U Trait Contribution P Value 12 292 C A GMSTP 0.9735360.000509 37 145 C A GMSTP 0.2769 0.000315 39 169 T A GMSTP 0.27214050.00034675 42 386 A G GMSTP 0.379311 0.000192 51 708 C A GMSTP 0.319890.000287 55 491 C G GMSTP 0.544187 0.0001335 56 428 G A GMSTP 0.3472530.000573

TABLE 8 Contributions of Favorable Alleles to Increased WaterOptimization Identified by TASSEL SEQ SNP ID NO. Position F U Trait PValue Contribution  1 428 A G YGSMN 0.0084 11.3070462  2 216 G T YGSMN0.000054594 4.15  3 506 A G YGSMN 0.0005806 2.187  4 818-821 — CGCGYGSMN 0.0035 14.5878562  5 254 G A YGSMN 0.00022694 1.6857 A G GMSTP5.62 × 10⁻⁷ 0.3725  6 186-188 G A YGSMN 0.007 6.8929277  7 526 A C YGSMN0.000211972 3.3256  7* 526 A C YGSMN 0.0026 7.3903226  8 615-616 — GAYGSMN 0.03 5.665  9 375 A G YGSMN 0.0143 6.5897654 10 331 A G YGSMN0.000435023 3.01738  10* 331 A G YGSMN 0.0026 7.3903226 11 210 A G GMSTP0.2431269 0.599 12 292 C A YGSMN 0.0031 4.2222 13 166 A G YGSMN 0.02635.4031514 A G GMSTP 0.000204245 1.1278 15  94 C G YGSMN 4.38 × 10⁻⁶1.3181  15*  94 C G YGSMN 0.0309 7.624524 16  35 A T GMSTP 0.000956460.086 17 146 C A YGSMN 0.0071 8.327576 18 149 G C YGSMN 0.0006507941.312 19 432 G A GMSTP 5.47 × 10⁻¹⁵ 0.0393 20 753 A G YGSMN 0.00252.1981 21 755 G A YGSMN 0.000486298 2.2198 22 431 G C GMSTP 5.43 × 10⁻⁶0.4939 23 518 G T GMSTP 7.35 × 10⁻⁵ 1.2629 24 387 C G GMSTP 0.000397660.4522 25 660 A G GMSTP 0.00039306 0.4219 26 536 T C YGSMN 0.0007409460.7923 27 773-776 C G YGSMN 0.000138841 0.9736 C G GMSTP 0.0001247190.7974 28 310 T A YGSMN 1.87 × 10⁻⁷ 1.433 29 211 G A GMSTP 0.000340280.5831 30 401 G A YGSMN 0.0102 6.4804254 G A GMSTP 0.000177776 0.6844 31254 A G YGSMN 0.0044 8.7386037 G A GMSTP 0.00125 1.8112 32 439 A G YGSMN0.025 5.136 33 384 G A YGSMN 0.015 6.284 35 239 G A YGSMN 0.04954.6259439 G A GMSTP 0.000154145 1.4141 36 208 G A YGSMN 0.0002498751.7148 37 145 C A YGSMN 0.00029249 3.16538 38 535 A T GMSTP 0.0001802090.1236 39 169 T A GMSTP 0.000124333 1.2461 40  76 G A YGSMN 0.001211.9039947 41 724 A G YGSMN 2.71 × 10⁻⁵ 4.65472 42 386 A G YGSMN 0.003711.255257 43 375 A G GMSTP 0.000221511 0.6653 44 309 C G GMSTP 0.00110.1152 45 342 A C GMSTP 0.266801841 0.8445 46 445 G C YGSMN 0.0000328211.6764 47 602 A T YGSMN 0.000769319 3.7163 48 190 G A YGSMN 0.0002973083.369  48* 190 G A YGSMN 0.0054 8.0700349 49 593 C G YGSMN 0.00114283610.5852  49* 593 C G YGSMN 0.0282 7.642183 50 266-267 — AA YGSMN 0.0176.724 51 708 A C YGSMN 0.0054 7.3294598 C A GMSTP 2.42 × 10⁻⁵ 0.3221 52648 G A YGSMN 0.0026 10.9837972 53 541 A T YGSMN 0.0003 10.3325637 54442 C G YGSMN 0.000013938 11.0737 55 491 C G YGSMN 0.000238135 7.1354 55* 491 C G YGSMN 0.0446 9.1504902 56 428 A G YGSMN 0.000578625 0.702457 126 A G YGSMN 6.83 × 10⁻⁵ 3.70653 A G GMSTP 6.19 × 10⁻⁵ 0.5342*Values relate to tests of hybrids

TABLE 9 Contributions of Favorable Alleles to Increased WaterOptimization Identified by QIPDT2 SNP SEQ ID NO. Position F U TraitContribution P Value 12 292 C A GMSTP 0.973536 0.000509 14 148 G T GMSTP0.739413 0.000003 23 518 T G GWTPN 3.438703 0.000198 37 145 C A GMSTP0.2769 0.000315 39 169 T A GMSTP 0.2721405 0.00034675 42 386 A G GMSTP0.379311 0.000192 46 445 C G GMSTP 0.777738 0.000547 48 190 A G GMSTP1.47593 0.000274 51 708 C A GMSTP 0.31989 0.000287 52 648 G A GMSTP0.450848 0.000111 55 491 C G GMSTP 0.544187 0.0001335 56 428 G A GMSTP0.347253 0.000573

Materials and Methods Employed in Examples 8-12

NP2391 is an elite, non-Stiff Stalk variety of maize. NP2391 isdescribed in U.S. Pat. No. 7,166,783. NP2391 comprises a G allele atposition 87 of SEQ ID NO: 47, a G allele at position 386 of SEQ ID NO:46, a G allele at positions 4979-4981 of SEQ ID NO: 7, a C allele atposition 4641 of SEQ ID NO: 7, an A allele at position 472 of SEQ ID NO:48, a G allele at position 237 of SEQ ID NO: 56, an A allele at position516 of SEQ ID NO: 56, an A allele at position 266 of SEQ ID NO: 44, a Tallele at position 475 of SEQ ID NO: 45, a T allele at position 173 ofSEQ ID NO: 57, a C allele at position 746 of SEQ ID NO: 24, an A alleleat position 391 of SEQ ID NO: 33, a C allele at position 258 of SEQ IDNO: 29, an A allele at position 217 of SEQ ID NO: 23, a G allele atposition 116 of SEQ ID NO: 23, a G allele at position 463 of SEQ ID NO:19, a T allele at position 309 of SEQ ID NO: 19, a D allele at positions264-271 of SEQ ID NO: 2, an G allele at position 100 of SEQ ID NO: 2, aC allele at position 486 of SEQ ID NO: 58, a G allele at position 111 ofSEQ ID NO: 51, a G allele at position 254 of SEQ ID NO: 27, a G alleleat position 729 of SEQ ID NO: 59, a G allele at position 267 of SEQ IDNO: 60, a G allele at position 562 of SEQ ID NO: 25, a C allele atposition 1271 of SEQ ID NO: 26 and a G allele at position 193 of SEQ IDNO: 55. See FIG. 1.

NP2460 is an elite, Stiff Stalk variety of maize. NP2460 is described inU.S. Pat. No. 7,122,726. NP2460 comprises a C allele at position 386 ofSEQ ID NO: 46, an A allele at positions 4979-4981 of SEQ ID NO: 7, an Aallele at position 4641 of SEQ ID NO: 7, a G allele at position 472 ofSEQ ID NO: 48, a G allele at position 237 of SEQ ID NO: 56, a C alleleat position 516 of SEQ ID NO: 56, a C allele at position 266 of SEQ IDNO: 44, a C allele at position 475 of SEQ ID NO: 45, a G allele atposition 173 of SEQ ID NO: 57, a C allele at position 746 of SEQ ID NO:24, an A allele at position 391 of SEQ ID NO: 33, a T allele at position258 of SEQ ID NO: 29, an A allele at position 217 of SEQ ID NO: 23, a Gallele at position 116 of SEQ ID NO: 23, a C allele at position 463 ofSEQ ID NO: 19, a C allele at position 309 of SEQ ID NO: 19, a D alleleat positions 264-271 of SEQ ID NO: 2, an G allele at position 100 of SEQID NO: 2, a C allele at position 486 of SEQ ID NO: 58, a G allele atposition 254 of SEQ ID NO: 27, a G allele at position 729 of SEQ ID NO:59, a G allele at position 267 of SEQ ID NO: 60, a G allele at position562 of SEQ ID NO: 25, a C allele at position 1271 of SEQ ID NO: 26 andan A allele at position 193 of SEQ ID NO: 55. See FIG. 1.

CML333 is an exotic, inbred variety of maize from the InternationalMaize and Wheat Improvement Center (CIMMYT) in Mexico. CML333 is knownto be resistant to both southwestern corn borer and fall armyworm.CML333 comprises an A allele at positions 4979-4981 of SEQ ID NO: 7, a Gallele at position 472 of SEQ ID NO: 48, a G allele at position 237 ofSEQ ID NO: 56, a G allele at position 173 of SEQ ID NO: 57, a G alleleat 0172A, an A allele at position 116 of SEQ ID NO: 23, an A allele atposition 100 of SEQ ID NO: 2 and an A allele at position 267 of SEQ IDNO: 60. See FIG. 1.

CML322 is an exotic, inbred variety of maize from the InternationalMaize and Wheat Improvement Center (CIMMYT) in Mexico. CML322 comprisesa C allele at position 386 of SEQ ID NO: 46, an A allele at positions4979-4981 of SEQ ID NO: 7, a G allele at position 472 of SEQ ID NO: 48,a C allele at position 266 of SEQ ID NO: 44, a C allele at position 309of SEQ ID NO: 19, a C allele at position 111 of SEQ ID NO: 51, an Aallele at position 562 of SEQ ID NO: 25 and an A allele at position 1271of SEQ ID NO: 26. See FIG. 1.

Cateto SP VII is an exotic variety of maize that is native to Brazil.Although it demonstrates a high combining ability with many varieties ofmaize, it produces a relatively low yield. Cateto SP VII comprises an Aallele at position 87 of SEQ ID NO: 47, an A allele at position 4641 ofSEQ ID NO: 7, a G allele at position 472 of SEQ ID NO: 48, a C allele atposition 266 of SEQ ID NO: 44, an A allele at position 746 of SEQ ID NO:24, a T allele at position 258 of SEQ ID NO: 29, a G allele at position217 of SEQ ID NO: 23, an A allele at position 100 of SEQ ID NO: 2, an Aallele at position 486 of SEQ ID NO: 58 and an A allele at position 193of SEQ ID NO: 55. See FIG. 1.

Confite Morocho AYA 38 is an exotic, variety of maize that is native toPeru. Although it is resistant to Helminthosporium, it is susceptible torust. Confite morocho AYA 38 comprises an A allele at position 4641 ofSEQ ID NO: 7, a C allele at position 237 of SEQ ID NO: 56, a G allele atposition 391 of SEQ ID NO: 33, a C allele at position 309 of SEQ ID NO:19, an insertion at positions 264-271 of SEQ ID NO: 2 and an A allele atposition 486 of SEQ ID NO: 58. See FIG. 1.

Tuxpeno VEN 692 is an exotic, variety of maize that is native toVenezuela. It is highly resistant to both Helminthosporium and rust.Tuxpeno VEN 692 comprises an A allele at position 4641 of SEQ ID NO: 7,a C allele at position 237 of SEQ ID NO: 56, a G allele at position 258of SEQ ID NO: 29, a C allele at position 463 of SEQ ID NO: 19 and an Aallele at position 193 of SEQ ID NO: 55. See FIG. 1.

Maize plants of the NP2391 variety were crossed with 134 exoticvarieties of maize, including CML333, CML322, Cateto SP VII, ConfiteMorocho AYA 38, and Tuxpeno VEN 692. The progeny of these crosses werebackcrossed with NP2391 for five generations to create NP2391-exotichybrids. Segmental introgressions of the shaggy kinase gene (GENBANK®Database Accession No. AY103545; incorporated by reference herein) wereidentified in NP2391-exotic hybrids representing 42 of the exotic donors(CML333, CML322, Cateto SP VII, Confite Morocho AYA 38, Tuxpeno VEN 692,CML69, HH5982, TLT0766, CML103, M37W, TLZ0845, AGG742, NC358, P39,Serrano GUA 3, Mochero LBQ 17, KXI0970, 6B209, Cholito BOV 705, CML228,Coroico Amarillo, 8B006, EE8001, Enano M.D.3, Perola BOV 711, PuyaGrande SAN, XPRR001, B97, Cacao SAS 327, Tx303, Clavito ECU 366, EarlyCaribbean MAR 10, Patillo BOV 502, Rabo De Zorro ANC 325, Shajatu ANC120, Shoe Peg PI269743, St. Croix IVC 2, Oh7B, Polio VEN 336, Tzi8 andOh43). Each of the NP2391-exotic hybrids containing a segmentalintrogression in the shaggy kinase gene was selfed for two generations,selecting for progeny comprising the exotic donor genotype at positions4979-4981 of SEQ ID NO: 7/position 4641 of SEQ ID NO: 7. Two lines wereselected from the progeny of each NP2391-exotic hybrid selfing: onehomozygous for the exotic donor genotype at positions 4979-4981 of SEQID NO: 7/position 4641 of SEQ ID NO: 7 and the other homozygous for theNP2391 genotype at positions 4979-4981 of SEQ ID NO: 7/position 4641 ofSEQ ID NO: 7. Each of these lines was crossed with NP2460 to create anF1 hybrid line. In addition, NP2460 was crossed directly with NP2391 tocreate an F1 control hybrid.

The F1 hybrids were evaluated in four drought stress locations (LaSalle, Colo., United States of America; Gilroy, Calif., United States ofAmerica; Los Andes, Chile and Granaros, Chile), with six treatmentreplications at each location using a restricted, randomized blockdesign, as well as in twelve single-rep cornbelt locations. Droughtstress treatments were imposed around the time of pollination andconsisted of a period of water deficiency capable of decreasing grainyield by about 40-60%. The timing of each drought stress treatment wasdetermined by soil type and local climate (from which water-holdingcapacity and the evapotranspiration rate (ET) were estimated ormeasured). Normal irrigation was stopped about 3-4 weeks before mid-shedto allow the soil moisture level to drop to a critical level, whichlevel was reached approximately 7 days prior to pollination. Once thesoil dried to the critical level, deficit irrigation was commenced(approximately 40% of ET). Drought stress treatments were continued fortwo weeks following mid-shed, at which time normal irrigation wasresumed.

Drought tolerance was evaluated by measuring grain yield at standardmoisture percentage (YGSMN) and grain moisture at harvest (GMSTP). Thestatistical data quality control was done by plotting the distributionof the data. Residuals were generated using the following model:Y=μ+location+replications(location)+family+allele(family),wherein Y=dependent phenotype and μ=phenotypic average; Location andreplications (location) were random. Family and allele (family) werefixed. Residuals were analyzed across replications within a family andflagged according to specified criteria, which criteria depended uponthe trait being evaluated. The final analysis was performed for bothindividual locations and for locations combined using the followingmodel:Y=μ+replications+family+allele(family)+replications*family,wherein Y=dependent phenotype and μ=phenotypic average. Replicationswere random. Family and allele (family) were fixed. Least squares meanswere calculated and student's T test was run for pairwise comparisons.

As shown in Table 10, six of the F1 hybrids derived from crossing NP2460with a line homozygous for an exotic donor genotype at positions4979-4981 of SEQ ID NO: 7/position 4641 of SEQ ID NO: 7 demonstratedenhanced drought tolerance.

TABLE 10 Comparisons of NP2460 Hybrids with respect to SEQ ID NO: 7Difference under Yield (bu/ac) drought stress conditions (bu/ac) F1Hybrid Cornbelt Drought Stress vs. control hybrid vs. - hybrid Control180 162 CML333+ 184 176 14 * 11 * CML333− 179 165 CML322+ 185 182 20 *23 * CML322− 183 159 Cateto+ 214 168  6 * 18 * Cateto− 180 150 Confite+191 170  8 * 13 * Confite− 189 157 Tuxpeno+ 195 175 13 * 10 * Tuxpeno−159 165 * indicates p value < 0.05

Example 8 NP2460×(NP2391×CML333)

NP2391 was crossed with CML333, and progeny derived from that cross werebackcrossed with NP2391 for five generations to create an NP2391×CML333shaggy hybrid. The NP2391×CML333 shaggy hybrid was selfed for twogenerations, and two lines were selected based upon their genotype atpositions 4979-4981 of SEQ ID NO: 7: one line homozygous for the CML333genotype (AA) (“CML333 homozygous +”) and the other homozygous for theNP2931 genotype (GG) (“CML333 homozygous −”). See FIG. 2. Each of theselines was crossed with NP2460 to create an F1 hybrid. See FIG. 2.

As shown in Table 2, the F1 hybrid line created by crossing NP2460 withthe line comprising AA at positions 4979-4981 of SEQ ID NO: 7(“CML333+”) demonstrated enhanced drought tolerance as compared to boththe control hybrid and the F1 hybrid derived by crossing NP2460 with theline comprising GG at positions 4979-4981 of SEQ ID NO: 7 (“CML333−”).Under drought stress treatment, the CML333+ hybrid demonstrated asignificantly higher grain yield at standard moisture percentage (176bu/ac) than both the control hybrid (162 bu/ac) and the CML333− hybrid(165 bu/ac).

Example 9 NP2460×(NP2391×CML322)

NP2391 was crossed with CML322, and progeny derived from that cross werebackcrossed with NP2391 for five generations to create an NP2391×CML322shaggy hybrid. The NP2391×CML322 shaggy hybrid was selfed for twogenerations, and two lines were selected based upon their genotype atpositions 4979-4981 of SEQ ID NO: 7: one line homozygous for the CML322genotype (AA) (“CML322 homozygous +”) and the other homozygous for theNP2931 genotype (GG) (“CML322 homozygous −”). See FIG. 3. Each of theselines was crossed with NP2460 to create an F1 hybrid. See FIG. 3.

As shown in Table 2, the F1 hybrid line created by crossing NP2460 withthe line comprising AA at positions 4979-4981 of SEQ ID NO: 7(“CML322+”) demonstrated enhanced drought tolerance as compared to boththe control hybrid and the F1 hybrid derived by crossing NP2460 with theline comprising GG at positions 4979-4981 of SEQ ID NO: 7 (“CML322−”).Under drought stress treatment, the CML322+ hybrid demonstrated asignificantly higher grain yield at standard moisture percentage (182bu/ac) than both the control hybrid (162 bu/ac) and the CML322− hybrid(159 bu/ac). Notably, the grain yield of the CML322+ hybrid underdrought stress conditions was nearly identical to its yield undercornbelt conditions.

Example 10 NP2460×(NP2391× Cateto SP VII)

NP2391 was crossed with Cateto SP VII, and progeny derived from thatcross were backcrossed with NP2391 for five generations to create anNP2391× Cateto SP VII shaggy hybrid. The NP2391× Cateto SP VII shaggyhybrid was selfed for two generations, and two lines were selected basedupon their genotype at position 4641 of SEQ ID NO: 7: one linehomozygous for the Cateto SP VII genotype (AA) (“Cateto homozygous +”)and the other homozygous for the NP2931 genotype (CC) (“Catetohomozygous −”). See FIG. 4. Each of these lines was crossed with NP2460to create an F1 hybrid. See FIG. 4.

As shown in Table 2, the F1 hybrid line created by crossing NP2460 withthe line comprising AA at position 4641 of SEQ ID NO: 7 (“Cateto+”)demonstrated enhanced drought tolerance as compared to both the controlhybrid and the F1 hybrid derived by crossing NP2460 with the linecomprising CC at position 4641 of SEQ ID NO: 7 (“Cateto−”). Underdrought stress treatment, the Cateto+ hybrid demonstrated asignificantly higher grain yield at standard moisture percentage (168bu/ac) than both the control hybrid (162 bu/ac) and the Cateto− hybrid(150 bu/ac).

Example 11 NP2460×(NP2391× Confite Morocho AYA 38)

NP2391 was crossed with Confite Morocho AYA 38, and progeny derived fromthat cross were backcrossed with NP2391 for five generations to createan NP2391× Confite Morocho AYA 38 shaggy hybrid. The NP2391× ConfiteMorocho AYA 38 shaggy hybrid was selfed for two generations, and twolines were selected based upon their genotype at position 4641 of SEQ IDNO: 7: one line homozygous for the Confite Morocho AYA 38 genotype (AA)(“Confite homozygous +”) and the other homozygous for the NP2931genotype (CC) (“Confite homozygous −”). See FIG. 5. Each of these lineswas crossed with NP2460 to create an F1 hybrid. See FIG. 5.

As shown in Table 2, the F1 hybrid line created by crossing NP2460 withthe line comprising AA at position 4641 of SEQ ID NO: 7 (“Confite+”)demonstrated enhanced drought tolerance as compared to both the controlhybrid and the F1 hybrid derived by crossing NP2460 with the linecomprising CC at position 4641 of SEQ ID NO: 7 (“Confite−”). Underdrought stress treatment, the Confite+ hybrid demonstrated asignificantly higher grain yield at standard moisture percentage (170bu/ac) than both the control hybrid (162 bu/ac) and the Cateto− hybrid(157 bu/ac).

Example 12 NP2460×(NP2391× Tuxpeno VEN 692)

NP2391 was crossed with Tuxpeno VEN 692, and progeny derived from thatcross were backcrossed with NP2391 for five generations to create anNP2391× Tuxpeno VEN 692 shaggy hybrid. The NP2391× Tuxpeno VEN 692shaggy hybrid was selfed for two generations, and two lines wereselected based upon their genotype at position 4641 of SEQ ID NO: 7: oneline homozygous for the Tuxpeno VEN 692 genotype (AA) (“Tuxpenohomozygous +”) and the other homozygous for the NP2931 genotype (CC)(“Tuxpeno homozygous −”). See FIG. 7. Each of these lines was crossedwith NP2460 to create an F1 hybrid. See FIG. 7.

As shown in Table 2, the F1 hybrid line created by crossing NP2460 withthe line comprising AA at position 4641 of SEQ ID NO: 7 (“Tuxpeno+”)demonstrated enhanced drought tolerance as compared to both the controlhybrid and the F1 hybrid derived by crossing NP2460 with the linecomprising CC at position 4641 of SEQ ID NO: 7 (“Tuxpeno−”). Underdrought stress treatment, the Tuxpeno+ hybrid demonstrated asignificantly higher grain yield at standard moisture percentage (175bu/ac) than both the control hybrid (162 bu/ac) and the Tuxpeno− hybrid(165 bu/ac).

Example 13 Testing of Water Optimized Hybrids for Yield Gains

157 water optimized hybrids from 36 chasis were compared with basegenetic (control) plant in 9-21 locations, each under four environmenttypes: full irrigation, limited irrigation, non-irrigated(non-stress/receiving adequate rainfall), non-irrigated stress, dryland(low plant density; non-stress/receiving adequate rainfall) and drylandstress). As shown in Tables 11 and 12, water optimized hybridsoutperformed closely related base hybrids under all conditions tested.

TABLE 11 Yields Under Full Irrigation Conditions WO Base Hybrid HybridWO Base Haplotype* Haplotype Hybrid Hybrid Diff P Value ChIa CI 219.8205.4 14.3 0.108 aCGhI CGI 216.4 205.4 10.9 0.220 bCdeghil Cdeghi 224.5213.5 11.0 0.048 mean 220.2 208.1 12.1 SD 4.1 4.7 1.9 bCdefGhilCdefGh⁽¹⁾ 241.0 226.8 14.2 0.005 bCdefGhil CdefGh⁽²⁾ 238.0 226.8 11.20.030 CefGh cgi 204.4 192.1 12.3 0.077 CefGh cgi 211.2 192.1 19.2 0.018mean 223.7 209.4 14.2 SD 18.6 20.0 3.5 *Haplotype designations refer toHaplotypes A-M as set forth hereinabove. Uppercase letters indicate thatthe hybrid was homozygous for the corresponding haplotype, and lowercaseletters indicate the hybrid was heterozygous for the correspondinghaplotype. The absence of a letter from A-M in the designation indicatesthat the hybrid did not have that haplotype. ^((1),(2))indicate thatthese plants were derived from the same initial breeding but weredistinct individuals.

TABLE 12 Yields Under Limited Irrigation Conditions WO Base HybridHybrid WO Base Haplotype* Haplotype Hybrid Hybrid Diff P Value ChIa CI138.3 132.6 5.9 0.371 aCGhI CGI 145.9 132.6 13.3 0.039 bCdeghil Cdeghi149.2 142.1 7.2 0.266 mean 144.5 135.8 8.8 SD 5.5 5.5 4.0 *Haplotypedesignations are as in Table 11.

TABLE 13 Yields Under Dry Stress Conditions WO Base Hybrid Hybrid WOBase Haplotype* Haplotype Hybrid Hybrid Diff P Value bCdgil Cdgi 100.586.8 13.7 0.387 aCeGHI CeghI 107.5 88.5 19.0 0.178 aCeGHI CeghI 99.788.5 11.2 0.334 cdefGhijkl dfGh 109.3 90.9 18.4 0.157 cdefghl d 116.286.0 30.2 0.161 acghi cgi 102.2 81.5 20.7 0.066 acghi cgi 108.9 81.527.4 0.036 cefGhi cgi 96.9 77.7 19.2 0.100 cdfghe cd 95.2 81.6 13.60.201 mean 104.1 84.8 19.3 SD 6.8 4.4 6.3 *Haplotype designations are asin Table 11.

REFERENCES

All references listed below, as well as all references cited in theinstant disclosure, including but not limited to all patents, patentapplications and publications thereof, scientific journal articles, anddatabase entries (e.g., GENBANK® database entries and all annotationsavailable therein) are incorporated herein by reference in theirentireties to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, and/or compositionsemployed herein.

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

What is claimed is:
 1. A hybrid Zea mays plant having introgressed intoits genome a ZmDr4 water optimization locus that corresponds to SEQ IDNO: 67 [derived from maize variety CML322] that is associated withincreased yield at standard moisture percentage (YGSMN) and furtherwherein the water optimization locus comprises an AA dinucleotide at theposition that corresponds to positions 4979-4981 of SEQ ID NO:
 7. 2. TheZea mays plant of claim 1, wherein said introgression of the wateroptimization locus confers increased or stabilized yield in a waterstressed environment as compared to a control plant.
 3. The Zea maysplant of claim 1, wherein the plant is a elite Zea mays plant.
 4. TheZea mays plant of claim 1, wherein the water optimization locus furthercomprises at least one allele selected from the group consisting of: adeletion at positions 4497-4498 of SEQ ID NO: 7, a G nucleotide at theposition that corresponds to position 4505 of SEQ ID NO: 7, a Tnucleotide at the position that corresponds to position 4609 of SEQ IDNO: 7, an A nucleotide at the position that corresponds to position 4641of SEQ ID NO: 7, a T nucleotide at the position that corresponds toposition 4792 of SEQ ID NO: 7, a T nucleotide at the position thatcorresponds to position 4836 of SEQ ID NO: 7, a C nucleotide at theposition that corresponds to position 4844 of SEQ ID NO: 7, and a Gnucleotide at the position that corresponds to position 4969 of SEQ IDNO:
 7. 5. The Zea mays plant of claim 1, wherein the said plant furthercomprises in its genome any one of the following haplotypes selectedfrom the group consisting of: haplotype A, haplotype C, haplotype D,haplotype E, haplotype F, haplotype G, haplotype H, haplotype I,haplotype J, haplotype K, haplotype L, and haplotype M.
 6. The Zea maysplant of claim 1, wherein the plant is a Stiff Stalk maize variety or anon-Stiff Stalk maize variety.
 7. The Zea mays plant of claim 6, whereinthe plant is any one of NP2391, NP2460 or a progeny of NP2391 or NP2460.8. The Zea mays plant of claim 1, wherein the ZmDr4 locus comprises SEQID NO:
 7. 9. The Zea mays plant of claim 3, wherein the plant furthercomprises a transgene that encodes a gene product that providesresistance to a herbicide selected from among glyphosate, Sulfonylurea,imidazolinione, dicambia, glufisinate, phenoxy proprionic acid,cycloshexome, traizine, benzonitrile, and broxynil.
 10. A plant cell,plant seed or plant part derived from the plant of claim 1; wherein theplant cell, plant seed or plant part comprises said ZmDr4 wateroptimization locus.
 11. A progeny plant derived from the plant of claim1; wherein the progeny plant comprises said ZmDr4 water optimizationlocus.